vero cells  (Thermo Fisher)


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    Structured Review

    Thermo Fisher vero cells
    F protein of the neurovirulent A75/17-CDV is efficiently expressed, processed, and cell surface targeted, but it induces limited cell-cell fusion. (A) For F protein expression and processing analyses, Western blot analyses were performed from lysates of <t>Vero</t> cells <t>transfected</t> with the F protein expression plasmids as indicated. Twenty-four hours posttransfection, the lysates were separated by reducing SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The F proteins were revealed with an anti-F protein ectodomain serum. (B) Surface biotinylation of cells expressing different F proteins to determine F protein plasma membrane steady-state levels. Biotinylated proteins of F-protein-transfected (and derivative-transfected) cells were precipitated, and Western blot analyses were performed as indicated above. Black arrowheads highlight two unspecific bands precipitated in the assay and recognized by our anti-F protein serum. (C) Syncytium formation after cotransfection of the cells with plasmid DNA encoding CDV H-OP and F-A75/17, F-OP, or F-A75/17-ER. Mock-transfected cells (pCI) received CDV H-OP-encoding plasmid and empty vector; representative fields of view were photographed 24 h posttransfection. (D) For quantitative fusion assays, Vero cells either were infected with MVA-T7 (multiplicity of infection of 1) or were transfected with the different F proteins, a plasmid encoding H-OP, and a plasmid containing the CAT reporter gene under the control of the T7 promoter. Twelve hours after transfection, both cell populations were mixed and seeded into fresh plates. After 6 h at 37°C, fusion was quantified by measuring the amount of CAT protein produced. For each experiment, the value for the F-OP/H-OP combination was set to 100%. Means and standard deviations from three independent experiments performed in duplicate are shown.
    Vero Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Signal Peptide and Helical Bundle Domains of Virulent Canine Distemper Virus Fusion Protein Restrict Fusogenicity ▿"

    Article Title: Signal Peptide and Helical Bundle Domains of Virulent Canine Distemper Virus Fusion Protein Restrict Fusogenicity ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01287-07

    F protein of the neurovirulent A75/17-CDV is efficiently expressed, processed, and cell surface targeted, but it induces limited cell-cell fusion. (A) For F protein expression and processing analyses, Western blot analyses were performed from lysates of Vero cells transfected with the F protein expression plasmids as indicated. Twenty-four hours posttransfection, the lysates were separated by reducing SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The F proteins were revealed with an anti-F protein ectodomain serum. (B) Surface biotinylation of cells expressing different F proteins to determine F protein plasma membrane steady-state levels. Biotinylated proteins of F-protein-transfected (and derivative-transfected) cells were precipitated, and Western blot analyses were performed as indicated above. Black arrowheads highlight two unspecific bands precipitated in the assay and recognized by our anti-F protein serum. (C) Syncytium formation after cotransfection of the cells with plasmid DNA encoding CDV H-OP and F-A75/17, F-OP, or F-A75/17-ER. Mock-transfected cells (pCI) received CDV H-OP-encoding plasmid and empty vector; representative fields of view were photographed 24 h posttransfection. (D) For quantitative fusion assays, Vero cells either were infected with MVA-T7 (multiplicity of infection of 1) or were transfected with the different F proteins, a plasmid encoding H-OP, and a plasmid containing the CAT reporter gene under the control of the T7 promoter. Twelve hours after transfection, both cell populations were mixed and seeded into fresh plates. After 6 h at 37°C, fusion was quantified by measuring the amount of CAT protein produced. For each experiment, the value for the F-OP/H-OP combination was set to 100%. Means and standard deviations from three independent experiments performed in duplicate are shown.
    Figure Legend Snippet: F protein of the neurovirulent A75/17-CDV is efficiently expressed, processed, and cell surface targeted, but it induces limited cell-cell fusion. (A) For F protein expression and processing analyses, Western blot analyses were performed from lysates of Vero cells transfected with the F protein expression plasmids as indicated. Twenty-four hours posttransfection, the lysates were separated by reducing SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The F proteins were revealed with an anti-F protein ectodomain serum. (B) Surface biotinylation of cells expressing different F proteins to determine F protein plasma membrane steady-state levels. Biotinylated proteins of F-protein-transfected (and derivative-transfected) cells were precipitated, and Western blot analyses were performed as indicated above. Black arrowheads highlight two unspecific bands precipitated in the assay and recognized by our anti-F protein serum. (C) Syncytium formation after cotransfection of the cells with plasmid DNA encoding CDV H-OP and F-A75/17, F-OP, or F-A75/17-ER. Mock-transfected cells (pCI) received CDV H-OP-encoding plasmid and empty vector; representative fields of view were photographed 24 h posttransfection. (D) For quantitative fusion assays, Vero cells either were infected with MVA-T7 (multiplicity of infection of 1) or were transfected with the different F proteins, a plasmid encoding H-OP, and a plasmid containing the CAT reporter gene under the control of the T7 promoter. Twelve hours after transfection, both cell populations were mixed and seeded into fresh plates. After 6 h at 37°C, fusion was quantified by measuring the amount of CAT protein produced. For each experiment, the value for the F-OP/H-OP combination was set to 100%. Means and standard deviations from three independent experiments performed in duplicate are shown.

    Techniques Used: Expressing, Western Blot, Transfection, Polyacrylamide Gel Electrophoresis, Cotransfection, Plasmid Preparation, Infection, Produced

    2) Product Images from "Packaging Determinants in the UL11 Tegument Protein of Herpes Simplex Virus Type 1 ▿"

    Article Title: Packaging Determinants in the UL11 Tegument Protein of Herpes Simplex Virus Type 1 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01172-06

    Localization of UL11-GFP derivatives in HSV-1-infected cells. Vero cells transfected with the indicated constructs were infected with HSV-1 at 18 h posttransfection, and after an additional 15 to 20 h, the cells were visualized by confocal microscopy.
    Figure Legend Snippet: Localization of UL11-GFP derivatives in HSV-1-infected cells. Vero cells transfected with the indicated constructs were infected with HSV-1 at 18 h posttransfection, and after an additional 15 to 20 h, the cells were visualized by confocal microscopy.

    Techniques Used: Infection, Transfection, Construct, Confocal Microscopy

    UL11 packaging assay. (A) Vero cells transfected with wild-type UL11.GFP were infected with HSV-1 at 18 h posttransfection. After another 22 h, extracellular virions were purified from the medium by centrifugation through a 30% (wt/vol) sucrose cushion. Virion and infected-cell lysates were separated by SDS-PAGE in 12% gels and analyzed on Western blots using either anti-GFP serum (to detect UL11-GFP chimeras) or anti-VP16 serum (as a harvesting and loading control). (B) Packaging of a UL11 truncation mutant. Vero cells were transfected with the indicated UL11-GFP constructs or GFP alone and subsequently infected with HSV-1. The cell lysate and medium were then harvested and analyzed as described for panel A. (C) Packaging efficiency. Using densitometry, packaging efficiency was quantitated by dividing the amount of UL11-GFP protein in the medium (normalized for VP16) by the amount in the cell lysate (normalized for VP16). In each experiment, the wt UL11-GFP construct was set at 100% packaging efficiency. Shown is the average of four independent experiments.
    Figure Legend Snippet: UL11 packaging assay. (A) Vero cells transfected with wild-type UL11.GFP were infected with HSV-1 at 18 h posttransfection. After another 22 h, extracellular virions were purified from the medium by centrifugation through a 30% (wt/vol) sucrose cushion. Virion and infected-cell lysates were separated by SDS-PAGE in 12% gels and analyzed on Western blots using either anti-GFP serum (to detect UL11-GFP chimeras) or anti-VP16 serum (as a harvesting and loading control). (B) Packaging of a UL11 truncation mutant. Vero cells were transfected with the indicated UL11-GFP constructs or GFP alone and subsequently infected with HSV-1. The cell lysate and medium were then harvested and analyzed as described for panel A. (C) Packaging efficiency. Using densitometry, packaging efficiency was quantitated by dividing the amount of UL11-GFP protein in the medium (normalized for VP16) by the amount in the cell lysate (normalized for VP16). In each experiment, the wt UL11-GFP construct was set at 100% packaging efficiency. Shown is the average of four independent experiments.

    Techniques Used: Transfection, Infection, Purification, Centrifugation, SDS Page, Western Blot, Mutagenesis, Construct

    3) Product Images from "Characterization of the Lassa virus GP1 ectodomain shedding: implications for improved diagnostic platforms"

    Article Title: Characterization of the Lassa virus GP1 ectodomain shedding: implications for improved diagnostic platforms

    Journal: Virology Journal

    doi: 10.1186/1743-422X-6-147

    Intracellular expression and secretion of GP1 in cells transfected with intron-A containing and intronless LASV GPC constructs . (A) Intracellular expression of glycoprotein constructs [C]. The 42 KDa GP1 protein was readily detected in intron-containing GPC (lane 2), GP1-TM (lane 4), and sGP1-RRAA-FLAG (lane 5) cell extracts. Expression of GPC from an intronless construct resulted in significantly lower levels of detectable GP1 (lane 3). In vector control (lane 1) only endogenous proteins ca. 47 and 55 KDa were detected ( end. ). Relative average expression levels of each glycoprotein compared to intron-A containing GPC are indicated below the respective lanes, as estimated by densitometry. (B) Secreted GP1 was detected only in intron-A containing GPC and sGP1-RRAA-FLAG supernatants [S]. Relative level of secreted GP1 generated by sGP1-RRAA-FLAG compared to GPC, as estimated by densitometry, is indicated below lane 5B. (C) Immunoprecipitation of GP1 from GPC, GP1-TM, and sGP1-RRAA-FLAG transfected cell supernatants [IP]. Immunoprecipitated GP1 was detected in intron-A containing GPC (lane 2C) and sGP1-RRAA-FLAG (lane 5C), and at significantly lower levels in intronless GPC (lane 3C) expressing cell supernatants. A very faint GP1 band was detected in the immunoprecipitated GP1-TM sample (lane 4C). Vector control reactions did not show reactivity (lane 1C). Relative volume of each IP reaction loaded per lane is indicated above panel C. (D) Secreted GP1 profile in VERO cells transfected with LASV glycoprotein expression constructs. Average levels of GP1 from intronless GPC, GP1-TM, and sGP1-RRAA-FLAG, compared to those from intron-A containing GPC expression are shown below each lane in panels C and D (N = 4).
    Figure Legend Snippet: Intracellular expression and secretion of GP1 in cells transfected with intron-A containing and intronless LASV GPC constructs . (A) Intracellular expression of glycoprotein constructs [C]. The 42 KDa GP1 protein was readily detected in intron-containing GPC (lane 2), GP1-TM (lane 4), and sGP1-RRAA-FLAG (lane 5) cell extracts. Expression of GPC from an intronless construct resulted in significantly lower levels of detectable GP1 (lane 3). In vector control (lane 1) only endogenous proteins ca. 47 and 55 KDa were detected ( end. ). Relative average expression levels of each glycoprotein compared to intron-A containing GPC are indicated below the respective lanes, as estimated by densitometry. (B) Secreted GP1 was detected only in intron-A containing GPC and sGP1-RRAA-FLAG supernatants [S]. Relative level of secreted GP1 generated by sGP1-RRAA-FLAG compared to GPC, as estimated by densitometry, is indicated below lane 5B. (C) Immunoprecipitation of GP1 from GPC, GP1-TM, and sGP1-RRAA-FLAG transfected cell supernatants [IP]. Immunoprecipitated GP1 was detected in intron-A containing GPC (lane 2C) and sGP1-RRAA-FLAG (lane 5C), and at significantly lower levels in intronless GPC (lane 3C) expressing cell supernatants. A very faint GP1 band was detected in the immunoprecipitated GP1-TM sample (lane 4C). Vector control reactions did not show reactivity (lane 1C). Relative volume of each IP reaction loaded per lane is indicated above panel C. (D) Secreted GP1 profile in VERO cells transfected with LASV glycoprotein expression constructs. Average levels of GP1 from intronless GPC, GP1-TM, and sGP1-RRAA-FLAG, compared to those from intron-A containing GPC expression are shown below each lane in panels C and D (N = 4).

    Techniques Used: Expressing, Transfection, Gel Permeation Chromatography, Construct, Plasmid Preparation, Generated, Immunoprecipitation

    4) Product Images from "Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission"

    Article Title: Intercellular Extensions Are Induced by the Alphavirus Structural Proteins and Mediate Virus Transmission

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006061

    Alphavirus infection induces actin-positive intercellular extensions. (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see methods ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P
    Figure Legend Snippet: Alphavirus infection induces actin-positive intercellular extensions. (A) Vero cells were mock-transfected (Uninf.) or transfected with WT SINV or Y400K RNA, incubated at 37°C for 8 h, and fixed. Cells were permeabilized and stained with antibodies to detect the viral E2 envelope protein (red) and α-tubulin (blue), and with phalloidin to detect F-actin (green). Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments, which are quantitated in (B). Arrows indicate two examples of intercellular extensions; note that each is positive for all three markers and is in contact with a neighboring cell. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2, actin and tubulin and their contact with a neighboring cell (see methods ). Graph in B shows the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. ** P

    Techniques Used: Infection, Transfection, Incubation, Staining, Confocal Microscopy, Standard Deviation

    The alphavirus structural proteins induce the formation of intercellular extensions that preferentially target non-expressing cells. (A) Vero cells were transfected with plasmids encoding the SFV structural proteins with or without capsid and with or without the E2 Y399R mutation. At 24 h post-transfection the cells were fixed, permeabilized, immunostained for the SFV E2 protein and stained with phalloidin-Alexa568 to detect F-actin. Images were acquired by confocal microscopy and are representative of three independent experiments. Images from one optical section are shown. Bar = 20 μm. (B) Using the conditions described in (A), the number of intercellular extensions per E2-expressing cell was quantitated based on positive staining for actin and contact with a neighboring cell. The graph represents the mean and standard deviation of three independent experiments. *P
    Figure Legend Snippet: The alphavirus structural proteins induce the formation of intercellular extensions that preferentially target non-expressing cells. (A) Vero cells were transfected with plasmids encoding the SFV structural proteins with or without capsid and with or without the E2 Y399R mutation. At 24 h post-transfection the cells were fixed, permeabilized, immunostained for the SFV E2 protein and stained with phalloidin-Alexa568 to detect F-actin. Images were acquired by confocal microscopy and are representative of three independent experiments. Images from one optical section are shown. Bar = 20 μm. (B) Using the conditions described in (A), the number of intercellular extensions per E2-expressing cell was quantitated based on positive staining for actin and contact with a neighboring cell. The graph represents the mean and standard deviation of three independent experiments. *P

    Techniques Used: Expressing, Transfection, Mutagenesis, Staining, Confocal Microscopy, Standard Deviation

    SINV cell-cell transmission is independent of NRAMP in target or producer cells. (A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.
    Figure Legend Snippet: SINV cell-cell transmission is independent of NRAMP in target or producer cells. (A) Down-regulation of target cell NRAMP. Vero cells were transfected with WT SINV or SFV RNA and incubated at 37°C for 5 h (producer cells). Target Vero cells stably expressing the PM-GFP marker were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2, and then plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated for 19 h at 37°C in the continued presence of iron as indicated. The % infected target cells was quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control target cells (which was set to 1). (B) Down-regulation of producer cell NRAMP. Vero cells were pretreated as in Fig 6A to downregulate NRAMP2, transfected with WT SINV or SFV RNA, and incubated at 37°C for 5 h (producer cells). Uninfected Vero cells stably expressing PM-GFP (target cells) were then plated onto the infected cells, and the co-cultures were incubated for 19 h at 37°C in the continued presence of iron as indicated. Infection of target cells was quantitated as in Fig 6A. The graphs in A and B represent the mean and standard deviation of three independent experiments.

    Techniques Used: Transmission Assay, Transfection, Incubation, Stable Transfection, Expressing, Marker, Cell Culture, Infection, Staining, Standard Deviation

    Intercellular extensions are induced by various alphaviruses in Vero and primary human cells. (A) Vero cells were mock-infected (Uninf.), transfected with SINV Y400K RNA, or infected with WT SINV, SFV or CHIKV (MOI 20, 10, 10, respectively). Cells were then incubated at 37°C for 11 h, fixed, permeabilized, and stained with antibodies to detect viral envelope proteins (virus GP) and α-tubulin, and with phalloidin to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments. Bar = 20 μm. (B) Quantitation of the number of extensions in Vero cells from experiments as described in (A). Intercellular extensions emanating from infected cells were identified based on positive staining for GP, actin and tubulin and contact with a neighboring cell (see methods ). (C) HUVECs were infected and stained as in (A) and imaged and quantitated as in (B). Graphs in B and C show the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. * P
    Figure Legend Snippet: Intercellular extensions are induced by various alphaviruses in Vero and primary human cells. (A) Vero cells were mock-infected (Uninf.), transfected with SINV Y400K RNA, or infected with WT SINV, SFV or CHIKV (MOI 20, 10, 10, respectively). Cells were then incubated at 37°C for 11 h, fixed, permeabilized, and stained with antibodies to detect viral envelope proteins (virus GP) and α-tubulin, and with phalloidin to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of three independent experiments. Bar = 20 μm. (B) Quantitation of the number of extensions in Vero cells from experiments as described in (A). Intercellular extensions emanating from infected cells were identified based on positive staining for GP, actin and tubulin and contact with a neighboring cell (see methods ). (C) HUVECs were infected and stained as in (A) and imaged and quantitated as in (B). Graphs in B and C show the mean and standard deviation of three independent experiments, with 10 cells quantitated in each sample including the uninfected cells. * P

    Techniques Used: Infection, Transfection, Incubation, Staining, Confocal Microscopy, Quantitation Assay, Standard Deviation

    Formation of intercellular extensions does not require virus budding. Vero cells were mock-transfected (Uninf.) or transfected with SFV WT or G91D mutant RNA, incubated at the permissive temperature (28°C) overnight (A) or at the non-permissive temperature (37°C) for 8 h (B), and fixed. Note that different times of incubation at these two temperatures were used to allow virus replication and extension formation. Cells were permeabilized and immunostained to detect the virus glycoproteins (GP) or α-tubulin, and stained with phalloidin-Alexa488 to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (C,D) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for GP, actin and tubulin and their contact with a neighboring cell. (E,F) Vero cells were transfected with WT or G91D SFV RNA and incubated at 37°C for 2h (producer cells). At 4 h post-transfection Vero target cells stably expressing the PM-GFP marker were plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated overnight at 37 or 28°C. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in C-F represent the mean and standard deviation of three independent experiments. * P
    Figure Legend Snippet: Formation of intercellular extensions does not require virus budding. Vero cells were mock-transfected (Uninf.) or transfected with SFV WT or G91D mutant RNA, incubated at the permissive temperature (28°C) overnight (A) or at the non-permissive temperature (37°C) for 8 h (B), and fixed. Note that different times of incubation at these two temperatures were used to allow virus replication and extension formation. Cells were permeabilized and immunostained to detect the virus glycoproteins (GP) or α-tubulin, and stained with phalloidin-Alexa488 to detect F-actin. Cells were imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (C,D) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for GP, actin and tubulin and their contact with a neighboring cell. (E,F) Vero cells were transfected with WT or G91D SFV RNA and incubated at 37°C for 2h (producer cells). At 4 h post-transfection Vero target cells stably expressing the PM-GFP marker were plated onto the infected cells at an approximate ratio of 1:1 and the co-cultures incubated overnight at 37 or 28°C. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in C-F represent the mean and standard deviation of three independent experiments. * P

    Techniques Used: Transfection, Mutagenesis, Incubation, Staining, Confocal Microscopy, Infection, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

    Intercellular extensions do not transfer cytosolic or PM markers. (A) Vero cells were transfected with SINV WT-mCherry RNA (t = 0) and incubated for 6.5 h at 37°C. Cells were labeled for 30 min with CellTracker Green and then uninfected Vero cells were added and the co-cultures were incubated for 2 h at 37°C. Cells were then fixed and imaged by epifluorescence microscopy. Images are representative of three independent experiments, evaluating 10 cell pairs/experiment. Bar = 20 μm. (B) Vero cells expressing the PM-GFP marker were transfected with SINV WT-mCherry RNA and incubated at 37°C for 7 h. Uninfected Vero cells were then added and the co-cultures were incubated for 2 h at 37°C. Cells were fixed, stained with antibodies to detect α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown, and are representative of three independent experiments, 10 cell pairs/experiment. Bar = 20 μm.
    Figure Legend Snippet: Intercellular extensions do not transfer cytosolic or PM markers. (A) Vero cells were transfected with SINV WT-mCherry RNA (t = 0) and incubated for 6.5 h at 37°C. Cells were labeled for 30 min with CellTracker Green and then uninfected Vero cells were added and the co-cultures were incubated for 2 h at 37°C. Cells were then fixed and imaged by epifluorescence microscopy. Images are representative of three independent experiments, evaluating 10 cell pairs/experiment. Bar = 20 μm. (B) Vero cells expressing the PM-GFP marker were transfected with SINV WT-mCherry RNA and incubated at 37°C for 7 h. Uninfected Vero cells were then added and the co-cultures were incubated for 2 h at 37°C. Cells were fixed, stained with antibodies to detect α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown, and are representative of three independent experiments, 10 cell pairs/experiment. Bar = 20 μm.

    Techniques Used: Transfection, Incubation, Labeling, Epifluorescence Microscopy, Expressing, Marker, Staining, Confocal Microscopy

    Intercellular extensions are generated by stable cell-cell contacts and migration of infected cells, and contain budding virus particles. (A) Vero cells stably expressing GFP-actin were infected with WT-mCherry SINV and incubated for 7 h at 37°C. The cells were then imaged using the TIRF microscope in the wide-field mode and both the 561-nm and 488-nm lasers. Images were acquired every 10 s for 60 min (see S1 Movie ). Arrows indicate stable contacts established between an infected cell and a neighboring cell, and marker lines are used to follow cell position during migration. Bar = 20 μm. (B-E) Vero cells were infected with WT SINV, incubated at 37°C for 9 h, fixed, processed for SEM, and imaged using a Zeiss Supra 40 field emission SEM. (B) A representative image of infected cells. Bar = 10 μm. (C) SEM image of the region indicated by the dashed white box in panel B. Bar = 10 μm. (D and E) SEM images of the regions indicated by the dashed white boxes in panel C. Arrowheads indicate virus-sized structures. Bar = 1 μm.
    Figure Legend Snippet: Intercellular extensions are generated by stable cell-cell contacts and migration of infected cells, and contain budding virus particles. (A) Vero cells stably expressing GFP-actin were infected with WT-mCherry SINV and incubated for 7 h at 37°C. The cells were then imaged using the TIRF microscope in the wide-field mode and both the 561-nm and 488-nm lasers. Images were acquired every 10 s for 60 min (see S1 Movie ). Arrows indicate stable contacts established between an infected cell and a neighboring cell, and marker lines are used to follow cell position during migration. Bar = 20 μm. (B-E) Vero cells were infected with WT SINV, incubated at 37°C for 9 h, fixed, processed for SEM, and imaged using a Zeiss Supra 40 field emission SEM. (B) A representative image of infected cells. Bar = 10 μm. (C) SEM image of the region indicated by the dashed white box in panel B. Bar = 10 μm. (D and E) SEM images of the regions indicated by the dashed white boxes in panel C. Arrowheads indicate virus-sized structures. Bar = 1 μm.

    Techniques Used: Generated, Stable Transfection, Migration, Infection, Expressing, Incubation, Microscopy, Marker

    Formation of intercellular extensions and virus cell-cell transmission are independent of NRAMP2. (A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH 4 Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P
    Figure Legend Snippet: Formation of intercellular extensions and virus cell-cell transmission are independent of NRAMP2. (A) Effect of NRAMP downregulation on free virus infection. Vero cells were cultured for 3 days in control media or media containing 200 μg/ml ammonium iron citrate to down-regulate the SINV receptor NRAMP2. Cells were infected with SINV or SFV (MOI = 5) for 2 h at 37°C. 20 mM NH 4 Cl was then added to the medium to prevent secondary infection. Cells were fixed at 24 h post-infection and the ratio of infected to total cells quantitated by staining with antibody to the SINV or SFV E2 protein. The graph represents the mean and standard deviation of three independent experiments, with infection normalized to that of control cells (which was set to 1). * P

    Techniques Used: Transmission Assay, Infection, Cell Culture, Staining, Standard Deviation

    Production of fusion-active virus is required for cell-to-cell transmission. (A) Vero cells were transfected with SFV WT or D188K mutant RNA, incubated at 37°C and fixed at 8 hours post transfection. Cells were permeabilized and immunostained to detect the virus E2 glycoprotein or α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2 and tubulin and their contact with a neighboring cell. (C) Vero cells were transfected with WT or D188K SFV RNA and incubated at 37°C for 2 h (producer cells). At 4 hours post transfection Vero target cells stably expressing the PM-GFP marker were plated onto the producer cells at an approximate ratio of 1:1 and the co-cultures incubated at 37°C overnight. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in B and C represent the mean and standard deviation of three independent experiments. *** P
    Figure Legend Snippet: Production of fusion-active virus is required for cell-to-cell transmission. (A) Vero cells were transfected with SFV WT or D188K mutant RNA, incubated at 37°C and fixed at 8 hours post transfection. Cells were permeabilized and immunostained to detect the virus E2 glycoprotein or α-tubulin, and imaged by confocal microscopy. Images from one optical section are shown and are representative of the images from three independent experiments. Bar = 20 μm. (B) The number of intercellular extensions per infected cell (n = 10) was quantitated based on their positive staining for E2 and tubulin and their contact with a neighboring cell. (C) Vero cells were transfected with WT or D188K SFV RNA and incubated at 37°C for 2 h (producer cells). At 4 hours post transfection Vero target cells stably expressing the PM-GFP marker were plated onto the producer cells at an approximate ratio of 1:1 and the co-cultures incubated at 37°C overnight. Cells were then fixed, permeabilized, and immunostained to detect the viral glycoproteins. Epifluorescence microscopy was used to acquire 5 images using the 20X objective. The number of infected PM-GFP-positive target cells was quantitated and expressed as a fraction of the total number of target cells. The graphs in B and C represent the mean and standard deviation of three independent experiments. *** P

    Techniques Used: Transmission Assay, Transfection, Mutagenesis, Incubation, Confocal Microscopy, Infection, Staining, Stable Transfection, Expressing, Marker, Epifluorescence Microscopy, Standard Deviation

    5) Product Images from "Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses"

    Article Title: Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.109900

    Toxicity evaluation of the Cb AEs in Vero cells, A549 cells and ICR mice. (A, B) Cytotoxicity of Cb AEs to Vero cells (A) or A549 cells (B) was measured by MTT assays. (C-D) Toxicity assay of Cb AEs in ICR mice. (C) Mortality rate of acute intravenous (i.v.) administration of the Cb AEs in ICR mice (n=4 per group). (D) Body weight during the 7-day monitoring period in ICR mice subjected to acute intranasal (i.n.) administration of the Cb AEs (n=4 per group).
    Figure Legend Snippet: Toxicity evaluation of the Cb AEs in Vero cells, A549 cells and ICR mice. (A, B) Cytotoxicity of Cb AEs to Vero cells (A) or A549 cells (B) was measured by MTT assays. (C-D) Toxicity assay of Cb AEs in ICR mice. (C) Mortality rate of acute intravenous (i.v.) administration of the Cb AEs in ICR mice (n=4 per group). (D) Body weight during the 7-day monitoring period in ICR mice subjected to acute intranasal (i.n.) administration of the Cb AEs (n=4 per group).

    Techniques Used: Mouse Assay, MTT Assay

    6) Product Images from "Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses"

    Article Title: Broad-spectrum virucidal activity of bacterial secreted lipases against flaviviruses, SARS-CoV-2 and other enveloped viruses

    Journal: bioRxiv

    doi: 10.1101/2020.05.22.109900

    Identification of Csp_BJ secreted antiviral effector(s) against DENV or ZIKV. (A) The protein description was obtained from the UniProt and NCBI databases. (B) The identified Csp_BJ proteins were expressed and purified from E. coli cells. (C, D) A total of 1 µg of purified recombinant protein was mixed with 50 pfu of DENV (C) or ZIKV (D) in VP-SFM medium and incubated for 1 hr before infecting Vero cell monolayers. (E-G) Csp_BJ inhibited DENV and ZIKV infections via its secreted proteins Cb AE-1 and Cb AE-2. (E) The identified Csp_BJ secreted proteins Cb AE-1 and Cb AE-2 were expressed and purified from E. coli cells. (F, G) Inhibition curves of Cb AE-1 and Cb AE-2 against DENV (F) and ZIKV (G). Serial concentrations of Cb AE-1 or Cb AE-2 were mixed with 50 pfu of DENV or ZIKV in VP-SFM medium to perform standard plaque reduction neutralization tests (PRNTs). (C, D) Significance was determined using unpaired t-tests. Data are presented as the mean ± SEM.
    Figure Legend Snippet: Identification of Csp_BJ secreted antiviral effector(s) against DENV or ZIKV. (A) The protein description was obtained from the UniProt and NCBI databases. (B) The identified Csp_BJ proteins were expressed and purified from E. coli cells. (C, D) A total of 1 µg of purified recombinant protein was mixed with 50 pfu of DENV (C) or ZIKV (D) in VP-SFM medium and incubated for 1 hr before infecting Vero cell monolayers. (E-G) Csp_BJ inhibited DENV and ZIKV infections via its secreted proteins Cb AE-1 and Cb AE-2. (E) The identified Csp_BJ secreted proteins Cb AE-1 and Cb AE-2 were expressed and purified from E. coli cells. (F, G) Inhibition curves of Cb AE-1 and Cb AE-2 against DENV (F) and ZIKV (G). Serial concentrations of Cb AE-1 or Cb AE-2 were mixed with 50 pfu of DENV or ZIKV in VP-SFM medium to perform standard plaque reduction neutralization tests (PRNTs). (C, D) Significance was determined using unpaired t-tests. Data are presented as the mean ± SEM.

    Techniques Used: Purification, Recombinant, Incubation, Inhibition, Neutralization

    The virucidal activity of Cb AEs is mediated by enzymatic degradation of the viral lipidic envelope. (A) Lipase enzymatic activity of Cb AE-1, Cb AE-2 and Cb AE-1-S187G measured via an egg yolk agar plate assay. (B, C) Analysis of the exposure of DENV (B) or ZIKV (C) genomic RNA. DENV or ZIKV was first treated with serial concentrations of Cb AE-1, Cb AE-2 or BSA and then with RNase-A. Viral RNA degradation was evaluated by RT-qPCR. (D, E) The S187G mutant of Cb AE-1 fully lost its ability to suppress DENV (D) and ZIKV (E) infection: inhibition curves of Cb AE-1 and Cb AE-1-S187G against DENV (D) or ZIKV(E). Serial concentrations of Cb AE-1 or Cb AE-1-S187G were mixed with 50 pfu of DENV or ZIKV in VP-SFM medium to perform standard plaque reduction neutralization tests (PRNTs). (F) Representative negative stained transmission electron microscopy images of ZIKV particles treated with 10 µg/ml BSA (arrow head) and those treated with 10 µg/ml Cb AE-1 (empty arrow head); high magnification: 120,000×, low magnification: 30,000×. (G, H) Rate of DENV (G) or ZIKV (H) replication inhibition following exposure to Cb AEs before viral infection of Vero cell monolayers. The viral genome was quantified by RT-qPCR. (B, C, G, H) Significance was determined using unpaired t-tests. Data are presented as the mean ± SEM.
    Figure Legend Snippet: The virucidal activity of Cb AEs is mediated by enzymatic degradation of the viral lipidic envelope. (A) Lipase enzymatic activity of Cb AE-1, Cb AE-2 and Cb AE-1-S187G measured via an egg yolk agar plate assay. (B, C) Analysis of the exposure of DENV (B) or ZIKV (C) genomic RNA. DENV or ZIKV was first treated with serial concentrations of Cb AE-1, Cb AE-2 or BSA and then with RNase-A. Viral RNA degradation was evaluated by RT-qPCR. (D, E) The S187G mutant of Cb AE-1 fully lost its ability to suppress DENV (D) and ZIKV (E) infection: inhibition curves of Cb AE-1 and Cb AE-1-S187G against DENV (D) or ZIKV(E). Serial concentrations of Cb AE-1 or Cb AE-1-S187G were mixed with 50 pfu of DENV or ZIKV in VP-SFM medium to perform standard plaque reduction neutralization tests (PRNTs). (F) Representative negative stained transmission electron microscopy images of ZIKV particles treated with 10 µg/ml BSA (arrow head) and those treated with 10 µg/ml Cb AE-1 (empty arrow head); high magnification: 120,000×, low magnification: 30,000×. (G, H) Rate of DENV (G) or ZIKV (H) replication inhibition following exposure to Cb AEs before viral infection of Vero cell monolayers. The viral genome was quantified by RT-qPCR. (B, C, G, H) Significance was determined using unpaired t-tests. Data are presented as the mean ± SEM.

    Techniques Used: Activity Assay, Quantitative RT-PCR, Mutagenesis, Infection, Inhibition, Neutralization, Staining, Transmission Assay, Electron Microscopy

    Secreted effector(s) from Csp_BJ inhibit DENV and ZIKV infection in Vero cells. (A-C) Secreted factor(s) from Csp_BJ inhibited DENV and ZIKV infection in Vero cells. (A) Schematic representation of the study design. A Csp_BJ suspension was separated into bacterial cells and culture supernatant by centrifugation. Either the bacterial lysates or the culture supernatant (50% v/v) mixed with 50 pfu of DENV or ZIKV in VP-SFM medium (50% v/v) were incubated for 1 hr before being used for infection of Vero cell monolayers. Vero cells infected with fresh LB broth mixed with virus-containing medium served as a negative control. (B, C) Inhibition rate in the presence of cell lysates or culture supernatant of Csp_BJ following infection by DENV (B) or ZIKV (C) in Vero cells, as determined by plaque formation assay. (D–F) Proteins in the supernatant inhibited DENV and ZIKV infection in Vero cells. (D) Schematic representation of the study design. Either the retentate (proteins) or the filtrate (small molecules and short peptides) (50% v/v) was mixed with 50 pfu of DENV or ZIKV in VP-SFM medium (50% v/v) and incubated for 1 hr before inoculation into Vero cell monolayers. Vero cells infected with fresh LB broth or culture supernatant mixed with virus-containing medium served as negative or positive controls, respectively. (E, F) The inhibition rate in Vero cells was determined by plaque formation assay. (B, C, E, F) Significance was determined by unpaired t-tests. Data are presented as the mean ± SEM.
    Figure Legend Snippet: Secreted effector(s) from Csp_BJ inhibit DENV and ZIKV infection in Vero cells. (A-C) Secreted factor(s) from Csp_BJ inhibited DENV and ZIKV infection in Vero cells. (A) Schematic representation of the study design. A Csp_BJ suspension was separated into bacterial cells and culture supernatant by centrifugation. Either the bacterial lysates or the culture supernatant (50% v/v) mixed with 50 pfu of DENV or ZIKV in VP-SFM medium (50% v/v) were incubated for 1 hr before being used for infection of Vero cell monolayers. Vero cells infected with fresh LB broth mixed with virus-containing medium served as a negative control. (B, C) Inhibition rate in the presence of cell lysates or culture supernatant of Csp_BJ following infection by DENV (B) or ZIKV (C) in Vero cells, as determined by plaque formation assay. (D–F) Proteins in the supernatant inhibited DENV and ZIKV infection in Vero cells. (D) Schematic representation of the study design. Either the retentate (proteins) or the filtrate (small molecules and short peptides) (50% v/v) was mixed with 50 pfu of DENV or ZIKV in VP-SFM medium (50% v/v) and incubated for 1 hr before inoculation into Vero cell monolayers. Vero cells infected with fresh LB broth or culture supernatant mixed with virus-containing medium served as negative or positive controls, respectively. (E, F) The inhibition rate in Vero cells was determined by plaque formation assay. (B, C, E, F) Significance was determined by unpaired t-tests. Data are presented as the mean ± SEM.

    Techniques Used: Infection, Centrifugation, Incubation, Negative Control, Inhibition, Plaque Formation Assay

    7) Product Images from "Development of improved vaccine cell lines against rotavirus"

    Article Title: Development of improved vaccine cell lines against rotavirus

    Journal: Scientific Data

    doi: 10.1038/sdata.2017.21

    RV replication in NEU2-KO Vero cells. Using the CRISPR-Cas system a functinal NUE2 gene was edited and the RV3, CDC-9 and Rotarix rotavirus strains were evaluated. QPCR was performed in triplicate and the value was normalized to 18s RNA and compared to wild type Vero cell control (wt ctrl). All samples were completed in triplicate ( n =3). The value presented here represents the mean of the triplicates±s.e.m.
    Figure Legend Snippet: RV replication in NEU2-KO Vero cells. Using the CRISPR-Cas system a functinal NUE2 gene was edited and the RV3, CDC-9 and Rotarix rotavirus strains were evaluated. QPCR was performed in triplicate and the value was normalized to 18s RNA and compared to wild type Vero cell control (wt ctrl). All samples were completed in triplicate ( n =3). The value presented here represents the mean of the triplicates±s.e.m.

    Techniques Used: CRISPR, Real-time Polymerase Chain Reaction

    Experimental workflow. This study included a tiered siRNA screening approach coupled with CRISPR-generated knockout Vero cell lines to evaluate host genes and their implication during RV infection. The primary siRNA screen was performed with pooled OTP-siRNA in MA104 cell line using the simian rotavirus strain RV3. Top hits were selected by a z-score analysis and subjected to deconvoluted validation siRNA screens in Vero cells with the rotavirus strains RV3-BB, Rotarix, CDC-9. Resulting hits were evaluated in CRISPR generated Vero cell lines with CDC-9 and Rotarix (GSK P5) strains.
    Figure Legend Snippet: Experimental workflow. This study included a tiered siRNA screening approach coupled with CRISPR-generated knockout Vero cell lines to evaluate host genes and their implication during RV infection. The primary siRNA screen was performed with pooled OTP-siRNA in MA104 cell line using the simian rotavirus strain RV3. Top hits were selected by a z-score analysis and subjected to deconvoluted validation siRNA screens in Vero cells with the rotavirus strains RV3-BB, Rotarix, CDC-9. Resulting hits were evaluated in CRISPR generated Vero cell lines with CDC-9 and Rotarix (GSK P5) strains.

    Techniques Used: CRISPR, Generated, Knock-Out, Infection

    8) Product Images from "Identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathways"

    Article Title: Identification of two mutation sites in spike and envelope proteins mediating optimal cellular infection of porcine epidemic diarrhea virus from different pathways

    Journal: Veterinary Research

    doi: 10.1186/s13567-017-0449-y

    The variation in the TM domain of E protein played a potential role in PEDV-host interaction. ( A ) Alignment of the amino acid sequences of the V4 region in E protein from DR13, ca-DR13, 85-7 parent, C30, C40, and E40 strains. C30, C40, and E40 shared the same variation, with a deletion of 16 LWLFV 20 and the L25P mutation. ( B ) Analysis of the putative TM domain of the PEDV parent and variant E protein with TMHMM Server v.2.0 [ 25 ]. The TM domain was predicted as I 15 to L 37 and I 10 to L 32 , respectively. ( C ) Comparative analysis of the mRNA levels of HSPA5 (encoding GRP78) and the protein production of GRP78 among the parent-, C30-, and C40-infected Vero cells. ( D , E ) Comparative analysis of the mRNA levels of the genes encoding IL-6 and IL-8 among the parent-, C30-, and C40-infected Vero cells by qPCR assay. The β-Actin gene served as an endogenous control. Error bars indicated the means of three independent experiments. ( F and G ) and ( H ) Comparative analysis of the mRNA levels of the genes encoding GRP78, IL-6, and IL-8 among the recombinant plasmids-transfected Vero cells by qPCR assay. The corresponding amount of empty plasmid (pIRES2-EGFP) was used as the mock control. ( I ) Comparison of the cell apoptosis level by flow cytometry with dual Annexin V-PI cell labeling. The infected cells were collected at 36 hpi, and the mock-infected cells were used as the control. Fluorescence-activated cell sorting was used to detect the fluorescent signals of Annexin V and PI, using channels FL-1 and FL-2, respectively. The figure was representative of two independent experiments. The graph representing the percentage of fluorescent signals of Annexin V and PI in each quadrant was shown on the left. ( J ) Comparative analysis of the levels of early apoptosis. C30, C40, and E40 showed higher early apoptosis levels than the 85-7 parent, A40, B40, and D40 strains.
    Figure Legend Snippet: The variation in the TM domain of E protein played a potential role in PEDV-host interaction. ( A ) Alignment of the amino acid sequences of the V4 region in E protein from DR13, ca-DR13, 85-7 parent, C30, C40, and E40 strains. C30, C40, and E40 shared the same variation, with a deletion of 16 LWLFV 20 and the L25P mutation. ( B ) Analysis of the putative TM domain of the PEDV parent and variant E protein with TMHMM Server v.2.0 [ 25 ]. The TM domain was predicted as I 15 to L 37 and I 10 to L 32 , respectively. ( C ) Comparative analysis of the mRNA levels of HSPA5 (encoding GRP78) and the protein production of GRP78 among the parent-, C30-, and C40-infected Vero cells. ( D , E ) Comparative analysis of the mRNA levels of the genes encoding IL-6 and IL-8 among the parent-, C30-, and C40-infected Vero cells by qPCR assay. The β-Actin gene served as an endogenous control. Error bars indicated the means of three independent experiments. ( F and G ) and ( H ) Comparative analysis of the mRNA levels of the genes encoding GRP78, IL-6, and IL-8 among the recombinant plasmids-transfected Vero cells by qPCR assay. The corresponding amount of empty plasmid (pIRES2-EGFP) was used as the mock control. ( I ) Comparison of the cell apoptosis level by flow cytometry with dual Annexin V-PI cell labeling. The infected cells were collected at 36 hpi, and the mock-infected cells were used as the control. Fluorescence-activated cell sorting was used to detect the fluorescent signals of Annexin V and PI, using channels FL-1 and FL-2, respectively. The figure was representative of two independent experiments. The graph representing the percentage of fluorescent signals of Annexin V and PI in each quadrant was shown on the left. ( J ) Comparative analysis of the levels of early apoptosis. C30, C40, and E40 showed higher early apoptosis levels than the 85-7 parent, A40, B40, and D40 strains.

    Techniques Used: Mutagenesis, Variant Assay, Infection, Real-time Polymerase Chain Reaction, Recombinant, Transfection, Plasmid Preparation, Flow Cytometry, Cytometry, Labeling, Fluorescence, FACS

    9) Product Images from "ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites"

    Article Title: ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites

    Journal: The EMBO Journal

    doi: 10.1038/emboj.2011.429

    2B, 2BC, 2C, 3A, and 3AB interact with ACBD3. ( A ) The mammalian two-hybrid assay. The indicated combination of a pACT construct and a pBIND construct was transfected into Vero cells together with pG5luc encoding a firefly luciferase. Cell lysates were
    Figure Legend Snippet: 2B, 2BC, 2C, 3A, and 3AB interact with ACBD3. ( A ) The mammalian two-hybrid assay. The indicated combination of a pACT construct and a pBIND construct was transfected into Vero cells together with pG5luc encoding a firefly luciferase. Cell lysates were

    Techniques Used: Two Hybrid Assay, Construct, Transfection, Luciferase

    10) Product Images from "A Conserved Peptide in West Nile Virus NS4A Protein Contributes to Proteolytic Processing and Is Essential for Replication ▿A Conserved Peptide in West Nile Virus NS4A Protein Contributes to Proteolytic Processing and Is Essential for Replication ▿ §"

    Article Title: A Conserved Peptide in West Nile Virus NS4A Protein Contributes to Proteolytic Processing and Is Essential for Replication ▿A Conserved Peptide in West Nile Virus NS4A Protein Contributes to Proteolytic Processing and Is Essential for Replication ▿ §

    Journal: Journal of Virology

    doi: 10.1128/JVI.05864-11

    Conservative mutations at E121 partially restore WNV KUN RNA replication and RC formation. Vero cells were transfected with the mutant pKUNrep4 plasmids and selected with 10 μg/ml puromycin for 6 days. Fixed cells were then labeled with either
    Figure Legend Snippet: Conservative mutations at E121 partially restore WNV KUN RNA replication and RC formation. Vero cells were transfected with the mutant pKUNrep4 plasmids and selected with 10 μg/ml puromycin for 6 days. Fixed cells were then labeled with either

    Techniques Used: Transfection, Mutagenesis, Labeling

    Ala substitutions within the PEPE motif prevent RC formation. Vero cells were transfected with the mutant pKUNrep4 plasmids and selected with 10 μg/ml puromycin for 6 days. Fixed cells were then labeled with either (A) NS3 (in green) and dsRNA
    Figure Legend Snippet: Ala substitutions within the PEPE motif prevent RC formation. Vero cells were transfected with the mutant pKUNrep4 plasmids and selected with 10 μg/ml puromycin for 6 days. Fixed cells were then labeled with either (A) NS3 (in green) and dsRNA

    Techniques Used: Transfection, Mutagenesis, Labeling

    11) Product Images from "MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism"

    Article Title: MicroRNA-sensitive Oncolytic Measles Viruses for Cancer-specific Vector Tropism

    Journal: Molecular Therapy

    doi: 10.1038/mt.2011.55

    miR7 regulates spreading, cytotoxicity, protein expression, and progeny virus production of MV-EGFP miR7 . ( a ) Vero cells were seeded in 6-well plates at a density of 6 × 10 5 cells/well. After attachment, cells were transfected with 0 or 20 nmol/l
    Figure Legend Snippet: miR7 regulates spreading, cytotoxicity, protein expression, and progeny virus production of MV-EGFP miR7 . ( a ) Vero cells were seeded in 6-well plates at a density of 6 × 10 5 cells/well. After attachment, cells were transfected with 0 or 20 nmol/l

    Techniques Used: Expressing, Transfection

    Syncytia formation and expression of the measles virus (MV) F gene containing microRNA-target sites are repressed by the presence of miR7 . ( a ) 2 × 10 5 Vero cells/well were seeded in 12-well plates and after attachment co-transfected with 0 or
    Figure Legend Snippet: Syncytia formation and expression of the measles virus (MV) F gene containing microRNA-target sites are repressed by the presence of miR7 . ( a ) 2 × 10 5 Vero cells/well were seeded in 12-well plates and after attachment co-transfected with 0 or

    Techniques Used: Expressing, Transfection

    12) Product Images from "Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus"

    Article Title: Trisaccharide containing α2,3-linked sialic acid is a receptor for mumps virus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1608383113

    Cell surface expression and fusion-supporting ability of WT and mutant HN proteins. ( A ) Expression of WT (pink) and mutant (green) HN proteins on transfected HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells was examined by flow cytometry. Mutants have an alanine substituted for the residue interacting with the third sugar from the nonreducing terminal. The empty plasmid served as the control (black). ( B ) NCI-H358 and IMR-32 cells transfected with expression plasmids encoding the MuV-HN protein (WT or Y369A), MuV-F protein, and EGFP were observed for syncytia formation using fluorescent microscopy at 1 or 2 d after transfection. (Scale bar: 200 µm.) The data are representative of three independently performed experiments.
    Figure Legend Snippet: Cell surface expression and fusion-supporting ability of WT and mutant HN proteins. ( A ) Expression of WT (pink) and mutant (green) HN proteins on transfected HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells was examined by flow cytometry. Mutants have an alanine substituted for the residue interacting with the third sugar from the nonreducing terminal. The empty plasmid served as the control (black). ( B ) NCI-H358 and IMR-32 cells transfected with expression plasmids encoding the MuV-HN protein (WT or Y369A), MuV-F protein, and EGFP were observed for syncytia formation using fluorescent microscopy at 1 or 2 d after transfection. (Scale bar: 200 µm.) The data are representative of three independently performed experiments.

    Techniques Used: Expressing, Mutagenesis, Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Microscopy

    Involvement of the third sugar from the nonreducing terminal in the HN protein–receptor interaction. Experiments were performed for the HN protein of MuV ( A ) and PIV5, PIV2, and SeV ( B ). ( Left ) Aromatic residues involved in the interaction with the third sugar from the nonreducing terminal (Glc-3 of SL) as well as residues stacked with them in solved (MuV and PIV5) or model HN protein structures (PIV2 and SeV) are shown in magenta. SLs (Sia-1, Gal-2, and Glc-3) are shown in cyan. ( Right ) HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells transfected with expression plasmids encoding the HN protein (WT or mutant), F protein, and EGFP were observed for syncytia formation using fluorescence microscopy at 2 d posttransfection. (Scale bar: 200 μm.)
    Figure Legend Snippet: Involvement of the third sugar from the nonreducing terminal in the HN protein–receptor interaction. Experiments were performed for the HN protein of MuV ( A ) and PIV5, PIV2, and SeV ( B ). ( Left ) Aromatic residues involved in the interaction with the third sugar from the nonreducing terminal (Glc-3 of SL) as well as residues stacked with them in solved (MuV and PIV5) or model HN protein structures (PIV2 and SeV) are shown in magenta. SLs (Sia-1, Gal-2, and Glc-3) are shown in cyan. ( Right ) HEK293 (MuV, PIV5, and PIV2) or Vero (SeV) cells transfected with expression plasmids encoding the HN protein (WT or mutant), F protein, and EGFP were observed for syncytia formation using fluorescence microscopy at 2 d posttransfection. (Scale bar: 200 μm.)

    Techniques Used: Gas Chromatography, Transfection, Expressing, Mutagenesis, Fluorescence, Microscopy

    13) Product Images from "Endoproteolytic Processing of the Lymphocytic Choriomeningitis Virus Glycoprotein by the Subtilase SKI-1/S1P"

    Article Title: Endoproteolytic Processing of the Lymphocytic Choriomeningitis Virus Glycoprotein by the Subtilase SKI-1/S1P

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.5.2866-2872.2003

    Effects of brefeldin A and monensin on the cleavage of GP-C of LCMV and Lassa virus. Vero cells transfected with a pCAGGS plasmid encoding either Lassa virus GP (lanes 2 and 3), LCMV GP (lanes 5, 6, and 7), or empty vector (lanes 1 and 4) were labeled with [ 35 S]methionine/[ 35 S]cysteine at 24 h after transfection for 30 min, followed by a 3-h chase. GP-C and GP-2 were immunoprecipitated by using a tetra-His antibody and analyzed by SDS-PAGE and fluorography.
    Figure Legend Snippet: Effects of brefeldin A and monensin on the cleavage of GP-C of LCMV and Lassa virus. Vero cells transfected with a pCAGGS plasmid encoding either Lassa virus GP (lanes 2 and 3), LCMV GP (lanes 5, 6, and 7), or empty vector (lanes 1 and 4) were labeled with [ 35 S]methionine/[ 35 S]cysteine at 24 h after transfection for 30 min, followed by a 3-h chase. GP-C and GP-2 were immunoprecipitated by using a tetra-His antibody and analyzed by SDS-PAGE and fluorography.

    Techniques Used: Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page

    14) Product Images from "Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii"

    Article Title: Identification of Novel Small RNAs and Characterization of the 6S RNA of Coxiella burnetii

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100147

    Northern blots showing CbSRs up-regulated (≥2 fold) in host cells relative to ACCM2. RNA was isolated from SCVs (3 dpi) grown in ACCM2 (A) and in Vero host cells (V). Hybridizations were performed at high stringency using biotinylated oligonucleotide probes specific to each CbSR. 3 µg RNA was used for all lanes. Apparent sizes of the CbSRs, as calculated from the Northern blots, are indicated. (Note: intensity of bands is not comparable between panels, since exposure times for each panel have not been optimized).
    Figure Legend Snippet: Northern blots showing CbSRs up-regulated (≥2 fold) in host cells relative to ACCM2. RNA was isolated from SCVs (3 dpi) grown in ACCM2 (A) and in Vero host cells (V). Hybridizations were performed at high stringency using biotinylated oligonucleotide probes specific to each CbSR. 3 µg RNA was used for all lanes. Apparent sizes of the CbSRs, as calculated from the Northern blots, are indicated. (Note: intensity of bands is not comparable between panels, since exposure times for each panel have not been optimized).

    Techniques Used: Northern Blot, Isolation

    C. burnetii 6S RNA copies per genome over a 14-d infection period. A. Number of C. burnetii genomes over a period of 14 d in infected Vero cells, as determined by qPCR with a primer set specific to rpoS . Values on graph represent the means ± standard deviations of the results of 6 independent determinations. B. Average number of copies of C. burnetii 6S RNAs per genome over a 14-d infection of Vero cells. The number of 6S RNA copies was determined by qRT-PCR using primers specific for 6S RNA and 1 µg total RNA from each time point using the same source cultures as panel A. Values represent the means ± standard deviations of the results of 6 independent determinations. Asterisks denote a significant difference relative to the 0-d sample (p
    Figure Legend Snippet: C. burnetii 6S RNA copies per genome over a 14-d infection period. A. Number of C. burnetii genomes over a period of 14 d in infected Vero cells, as determined by qPCR with a primer set specific to rpoS . Values on graph represent the means ± standard deviations of the results of 6 independent determinations. B. Average number of copies of C. burnetii 6S RNAs per genome over a 14-d infection of Vero cells. The number of 6S RNA copies was determined by qRT-PCR using primers specific for 6S RNA and 1 µg total RNA from each time point using the same source cultures as panel A. Values represent the means ± standard deviations of the results of 6 independent determinations. Asterisks denote a significant difference relative to the 0-d sample (p

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    C. burnetii total RNA separated on a denaturing gel. RNA isolated from C. burnetii LCVs (3 dpi) and SCVs (14 dpi) grown in Vero host cells, separated on a denaturing 8 M urea 8% acrylamide gel stained with ethidium bromide (5 µg RNA per lane). Arrow indicates the position of 6S RNA at ∼200 nucleotides. The number of nucleotides in RNA size standards (Std) is indicated to the left.
    Figure Legend Snippet: C. burnetii total RNA separated on a denaturing gel. RNA isolated from C. burnetii LCVs (3 dpi) and SCVs (14 dpi) grown in Vero host cells, separated on a denaturing 8 M urea 8% acrylamide gel stained with ethidium bromide (5 µg RNA per lane). Arrow indicates the position of 6S RNA at ∼200 nucleotides. The number of nucleotides in RNA size standards (Std) is indicated to the left.

    Techniques Used: Isolation, Acrylamide Gel Assay, Staining

    Northern blots showing 6S RNA levels of C. burnetii . RNA was isolated from LCVs (3 dpi) and SCVs (SCV 14 , 14 dpi; SCV 21 , 21 dpi) grown in Vero host cells and ACCM2, respectively. Hybridizations were performed at high stringency using a 6S RNA-specific biotinylated oligonucleotide probe. 3 µg RNA was used for all lanes. The size of the signal is indicated to the left.
    Figure Legend Snippet: Northern blots showing 6S RNA levels of C. burnetii . RNA was isolated from LCVs (3 dpi) and SCVs (SCV 14 , 14 dpi; SCV 21 , 21 dpi) grown in Vero host cells and ACCM2, respectively. Hybridizations were performed at high stringency using a 6S RNA-specific biotinylated oligonucleotide probe. 3 µg RNA was used for all lanes. The size of the signal is indicated to the left.

    Techniques Used: Northern Blot, Isolation

    15) Product Images from "Existence of Transdominant and Potentiating Mutants of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein, Suggests that Levels of UL9 Protein May Be Regulated during Infection"

    Article Title: Existence of Transdominant and Potentiating Mutants of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein, Suggests that Levels of UL9 Protein May Be Regulated during Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.17.9639-9651.2003

    The N-terminal domain of UL9 is able to dimerize. Vero cells were transfected with UL9-NT-AU1 (panels a, b, and c), wild-type UL9 (panels d, e, and f), or both (panels g, h, and i) and stained with anti-AU1 and RH7 antibodies. Green (fluorescein) represents staining with anti-AU1 antibody. Red (Texas Red) represents staining with RH7 antibody. The arrowheads mark untransfected cells in the same field, which can be used as a reference for background staining. Panels c, f, and i are merged images of panels a and b, d and e, and g and h, respectively.
    Figure Legend Snippet: The N-terminal domain of UL9 is able to dimerize. Vero cells were transfected with UL9-NT-AU1 (panels a, b, and c), wild-type UL9 (panels d, e, and f), or both (panels g, h, and i) and stained with anti-AU1 and RH7 antibodies. Green (fluorescein) represents staining with anti-AU1 antibody. Red (Texas Red) represents staining with RH7 antibody. The arrowheads mark untransfected cells in the same field, which can be used as a reference for background staining. Panels c, f, and i are merged images of panels a and b, d and e, and g and h, respectively.

    Techniques Used: Transfection, Staining

    Presence of motif V (G354A) mutation by itself is not sufficient to confer the potentiating phenotype. (A) Diagram of UL9 truncations. The N-terminal domain of UL9 is depicted as an open box. The C-terminal domain of UL9 is depicted as a gray box. The UL9 helicase motifs are depicted as black boxes. An asterisk points the position of the G354A mutation. The black line below the UL9 gene depicts the UL9-MV N-terminal fragment seen by Western blot analysis. (B) Plaque reduction assays with the indicated UL9 truncations were performed as described in Materials and Methods. The UL9-MV plasmid was used as a representative of a potentiating mutant. EP, empty plasmid; NP no plasmid. The error bars reflect the standard deviation calculated from at least three independent experiments. (C) The 17B antibody was used to detect UL9 truncations in concentrated lysates from Vero cells transfected with the plasmids used in the plaque reduction assay, described for panel A. Broad Range prestained protein markers are depicted on the right.
    Figure Legend Snippet: Presence of motif V (G354A) mutation by itself is not sufficient to confer the potentiating phenotype. (A) Diagram of UL9 truncations. The N-terminal domain of UL9 is depicted as an open box. The C-terminal domain of UL9 is depicted as a gray box. The UL9 helicase motifs are depicted as black boxes. An asterisk points the position of the G354A mutation. The black line below the UL9 gene depicts the UL9-MV N-terminal fragment seen by Western blot analysis. (B) Plaque reduction assays with the indicated UL9 truncations were performed as described in Materials and Methods. The UL9-MV plasmid was used as a representative of a potentiating mutant. EP, empty plasmid; NP no plasmid. The error bars reflect the standard deviation calculated from at least three independent experiments. (C) The 17B antibody was used to detect UL9 truncations in concentrated lysates from Vero cells transfected with the plasmids used in the plaque reduction assay, described for panel A. Broad Range prestained protein markers are depicted on the right.

    Techniques Used: Mutagenesis, Western Blot, Plasmid Preparation, Standard Deviation, Transfection

    Cotransfection of UL9-MV with wild-type UL9 results in enhanced nuclear localization. Vero cells were transfected with UL9-MV (a, b, and c), AU1-wild-type UL9 (d, e, and f), or both (g, h, and i) and stained with 17B (anti-UL9-NT) and RH7 (anti-UL9-CT) antibodies. Green (fluorescein) represents staining with 17B antibody. Red (Texas Red) represents staining with RH7 antibody. The arrowheads mark untransfected cells in the same field, which can be used as a reference for background staining. Panels c, f, and i are merged images of panels a and b, d and e, and g and h, respectively. Panels j and k contain images of Vero cells transfected with AU1-WT-UL9 and stained with anti-AU1 and RH7 antibodies. Green (fluorescein) represents staining with anti-AU1 antibody. Red (Texas Red) represents staining with RH7 antibody.
    Figure Legend Snippet: Cotransfection of UL9-MV with wild-type UL9 results in enhanced nuclear localization. Vero cells were transfected with UL9-MV (a, b, and c), AU1-wild-type UL9 (d, e, and f), or both (g, h, and i) and stained with 17B (anti-UL9-NT) and RH7 (anti-UL9-CT) antibodies. Green (fluorescein) represents staining with 17B antibody. Red (Texas Red) represents staining with RH7 antibody. The arrowheads mark untransfected cells in the same field, which can be used as a reference for background staining. Panels c, f, and i are merged images of panels a and b, d and e, and g and h, respectively. Panels j and k contain images of Vero cells transfected with AU1-WT-UL9 and stained with anti-AU1 and RH7 antibodies. Green (fluorescein) represents staining with anti-AU1 antibody. Red (Texas Red) represents staining with RH7 antibody.

    Techniques Used: Cotransfection, Transfection, Staining

    16) Product Images from "Porcine epidemic diarrhea virus nucleoprotein contributes to HMGB1 transcription and release by interacting with C/EBP-β"

    Article Title: Porcine epidemic diarrhea virus nucleoprotein contributes to HMGB1 transcription and release by interacting with C/EBP-β

    Journal: Oncotarget

    doi: 10.18632/oncotarget.11991

    PEDV-N enriches in the HMGB1 promotor and C/EBP binding motif is responsible for HMGB1 transcription A. The optimized sonication time in CHIP experiment was explored. B. Vero cells were infected with PEDV (MOI=0.1) for 24h. PCR analysis of ChIP samples demonstrated N protein enriched in the HMGB1 gene. C. Vero cells were transfected with PCA-N for 24h. PCR analysis of ChIP samples showed HA-tagged PEDV-N protein enriched in the HMGB1 gene. D. The reporter gene plasmids containing different HMGB1 promotor region were constructed, including pHLuc-HMGB1 (pHLuc1): −1622 to +83, pHLuc-HMGB1-1 (pHLuc2): −1264 to +83, pHLuc-MGB1-2 (pHLuc3): −822 to +83, and pHLuc-HMGB1-3 (pHLuc4): −382 to +83. Vero cells were cotransfected with each reporter gene plasmid and PCA-N, together with renilla luciferase reporter plasmid pRL-TK for 24h. Empty pGL3-basic and PCA vectors were used as the negative control. Luciferase activity was determined by a dual-luciferase assay system. E. Five mutant reporter gene plasmids with transcription factor binding site deletion were constructed, including mLuc2-C for C/EBP, mLuc2-M for v-Myb, mLuc2-AP for AP-1, mLuc2-Cd for CdxA, and mLuc2-U for USF. Vero cells were co-transfected with each mutant reporter gene or WT plasmid (pHLuc2), PCA-N or PCA and renilla luciferase reporter plasmid pRL-TK for 24h. Luciferase activity was determined. Data represented relative firefly luciferase activity normalized to renilla luciferase activity. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P
    Figure Legend Snippet: PEDV-N enriches in the HMGB1 promotor and C/EBP binding motif is responsible for HMGB1 transcription A. The optimized sonication time in CHIP experiment was explored. B. Vero cells were infected with PEDV (MOI=0.1) for 24h. PCR analysis of ChIP samples demonstrated N protein enriched in the HMGB1 gene. C. Vero cells were transfected with PCA-N for 24h. PCR analysis of ChIP samples showed HA-tagged PEDV-N protein enriched in the HMGB1 gene. D. The reporter gene plasmids containing different HMGB1 promotor region were constructed, including pHLuc-HMGB1 (pHLuc1): −1622 to +83, pHLuc-HMGB1-1 (pHLuc2): −1264 to +83, pHLuc-MGB1-2 (pHLuc3): −822 to +83, and pHLuc-HMGB1-3 (pHLuc4): −382 to +83. Vero cells were cotransfected with each reporter gene plasmid and PCA-N, together with renilla luciferase reporter plasmid pRL-TK for 24h. Empty pGL3-basic and PCA vectors were used as the negative control. Luciferase activity was determined by a dual-luciferase assay system. E. Five mutant reporter gene plasmids with transcription factor binding site deletion were constructed, including mLuc2-C for C/EBP, mLuc2-M for v-Myb, mLuc2-AP for AP-1, mLuc2-Cd for CdxA, and mLuc2-U for USF. Vero cells were co-transfected with each mutant reporter gene or WT plasmid (pHLuc2), PCA-N or PCA and renilla luciferase reporter plasmid pRL-TK for 24h. Luciferase activity was determined. Data represented relative firefly luciferase activity normalized to renilla luciferase activity. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P

    Techniques Used: Binding Assay, Sonication, Chromatin Immunoprecipitation, Infection, Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase, Negative Control, Activity Assay, Mutagenesis

    Blockage of HMGB1 inhibits PEDV infection A. Vero cells were infected with PEDV (MOI=0.1) and then treated with different concentrations HMGB1 antibody for 24h. The PEDV-N was analyzed by Western blot. B. The RNA level of viral ORF3 gene was evaluated in infected cells treated with HMGB1 antibody by qRT-PCR. C. The mRNA levels of proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) in infected cells treated with HMGB1 antibody treatment were evaluated by qRT-PCR. D. Vero cells were transfected with siHMGB1 to knockdown HMGB1 expression for 24h. siNC was used as the negative control. The cells were infected PEDV for 24h. The protein levels of HMGB1 and PEDV-N were analyzed by western blot. E. and F. The RNA levels of HMGB1 and PEDV ORF3 genes were measured by qRT-PCR. G. The mRNA levels of proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) in infected cells were evaluated by qRT-PCR after HMGB1 expression was knocked down. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P
    Figure Legend Snippet: Blockage of HMGB1 inhibits PEDV infection A. Vero cells were infected with PEDV (MOI=0.1) and then treated with different concentrations HMGB1 antibody for 24h. The PEDV-N was analyzed by Western blot. B. The RNA level of viral ORF3 gene was evaluated in infected cells treated with HMGB1 antibody by qRT-PCR. C. The mRNA levels of proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) in infected cells treated with HMGB1 antibody treatment were evaluated by qRT-PCR. D. Vero cells were transfected with siHMGB1 to knockdown HMGB1 expression for 24h. siNC was used as the negative control. The cells were infected PEDV for 24h. The protein levels of HMGB1 and PEDV-N were analyzed by western blot. E. and F. The RNA levels of HMGB1 and PEDV ORF3 genes were measured by qRT-PCR. G. The mRNA levels of proinflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) in infected cells were evaluated by qRT-PCR after HMGB1 expression was knocked down. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P

    Techniques Used: Infection, Western Blot, Quantitative RT-PCR, Transfection, Expressing, Negative Control

    PEDV-N promotes the acetylation/release of HMGB1 and expression of proinflammatory cytokines The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. A. Vero cells were infected with UV-inactivated PEDV (UV-PEDV) at different MOI for 1h at 37°C. Total and acetylated HMGB1 in the supernatant were then immediately analyzed by western blot. B. Vero cells were infected with PEDV (MOI=0.1). PEDV-N in cells was analyzed by western blot at different time points of the infection. C. Vero cells were transfected with PCA-N or PCA (2.5μg) for 12h, 24h, 36h. The mRNA level of HMGB1 or PEDV-N was analyzed by qRT-PCR. D. Vero cells were transfected with PCA-N or PCA at different doses (0.5, 1μg) for 36h. The mRNA of HMGB1 or N was analyzed. E. and F. The acetylated and total HMGB1 in the supernatant were determined by western blot in the cells transfected with PCA-N or PCA at the different amount or time points. The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. G. to I. Vero cells were first transfected with PCA-N or PCA at the same doses (2.5μg) for 12h and then treated with GAR, RES, or BAY for 24h. The acetylated HMGB1 and total HMGB1 in the supernatant were determined by western blot. J. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h, 24h, 36h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. K. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h. The cells were incubated with 1.5μg HMGB1 antibody for 24h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P
    Figure Legend Snippet: PEDV-N promotes the acetylation/release of HMGB1 and expression of proinflammatory cytokines The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. A. Vero cells were infected with UV-inactivated PEDV (UV-PEDV) at different MOI for 1h at 37°C. Total and acetylated HMGB1 in the supernatant were then immediately analyzed by western blot. B. Vero cells were infected with PEDV (MOI=0.1). PEDV-N in cells was analyzed by western blot at different time points of the infection. C. Vero cells were transfected with PCA-N or PCA (2.5μg) for 12h, 24h, 36h. The mRNA level of HMGB1 or PEDV-N was analyzed by qRT-PCR. D. Vero cells were transfected with PCA-N or PCA at different doses (0.5, 1μg) for 36h. The mRNA of HMGB1 or N was analyzed. E. and F. The acetylated and total HMGB1 in the supernatant were determined by western blot in the cells transfected with PCA-N or PCA at the different amount or time points. The protein concentration in the supernatant was determined using the Bradford assay. An equal amount of protein was used for western blot analysis. G. to I. Vero cells were first transfected with PCA-N or PCA at the same doses (2.5μg) for 12h and then treated with GAR, RES, or BAY for 24h. The acetylated HMGB1 and total HMGB1 in the supernatant were determined by western blot. J. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h, 24h, 36h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. K. Vero cells were transfected with PCA-N or PCA at the same doses (2.5μg) for 12h. The cells were incubated with 1.5μg HMGB1 antibody for 24h. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P

    Techniques Used: Expressing, Protein Concentration, Bradford Assay, Western Blot, Infection, Transfection, Quantitative RT-PCR, Incubation

    PEDV infection induces the increase of proinflammatory cytokines Vero cells were infected with PEDV (MOI=0.1). The cells were collected at 0, 2, 6, 12, 24hpi. A. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. B. The mRNA levels of HMGB1 were evaluated by qRT-PCR. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P
    Figure Legend Snippet: PEDV infection induces the increase of proinflammatory cytokines Vero cells were infected with PEDV (MOI=0.1). The cells were collected at 0, 2, 6, 12, 24hpi. A. The mRNA levels of proinflammatory cytokines were analyzed by qRT-PCR. B. The mRNA levels of HMGB1 were evaluated by qRT-PCR. The results are the representative of at least two different experiments. The results represent the means ±SD of triplicate determinations. One-way ANOVA; *, P

    Techniques Used: Infection, Quantitative RT-PCR

    17) Product Images from "Protection of IFNAR (-/-) Mice against Bluetongue Virus Serotype 8, by Heterologous (DNA/rMVA) and Homologous (rMVA/rMVA) Vaccination, Expressing Outer-Capsid Protein VP2"

    Article Title: Protection of IFNAR (-/-) Mice against Bluetongue Virus Serotype 8, by Heterologous (DNA/rMVA) and Homologous (rMVA/rMVA) Vaccination, Expressing Outer-Capsid Protein VP2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060574

    Expression of recombinant BTV proteins from rMVAs and pCi-neo plasmids by immunofluorescence. CEF cells infected with rMVA-VP2, VP5 or VP7, or Vero cells transfected with pCI-neo VP2, VP5 or VP7 were analysis by immunofluorescence assay. Empty MVA and pCI-neo were used as negative controls. Fluorescence was observed on cells using a sheep serum anti BTV-8 followed by Alexa Fluor 488-conjugated donkey anti-sheep IgG. Nuclei were staining with DAPI. BTV protein expression from pCI-neo plasmids or rMVAs encoding VP2, VP5 and VP7 proteins was observed by confocal microscopy.
    Figure Legend Snippet: Expression of recombinant BTV proteins from rMVAs and pCi-neo plasmids by immunofluorescence. CEF cells infected with rMVA-VP2, VP5 or VP7, or Vero cells transfected with pCI-neo VP2, VP5 or VP7 were analysis by immunofluorescence assay. Empty MVA and pCI-neo were used as negative controls. Fluorescence was observed on cells using a sheep serum anti BTV-8 followed by Alexa Fluor 488-conjugated donkey anti-sheep IgG. Nuclei were staining with DAPI. BTV protein expression from pCI-neo plasmids or rMVAs encoding VP2, VP5 and VP7 proteins was observed by confocal microscopy.

    Techniques Used: Expressing, Recombinant, Immunofluorescence, Infection, Transfection, Fluorescence, Staining, Confocal Microscopy

    18) Product Images from "Mitochondria Localize to the Cleavage Furrow in Mammalian Cytokinesis"

    Article Title: Mitochondria Localize to the Cleavage Furrow in Mammalian Cytokinesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0072886

    Mitochondria localize to the cleavage furrow in C2C12, Vero and Ptk2 cells. Fixed images of dividing C2C12, Vero and Ptk2 cells in early cytokinesis (C2C12), mid cytokinesis (Vero) and late cytokinesis (Ptk2). Cells were co-stained for mitochondria (MitoTracker Red, red), actin (Phallodin-488, green) and DNA (DAPI, blue). Bar, 10 µm.
    Figure Legend Snippet: Mitochondria localize to the cleavage furrow in C2C12, Vero and Ptk2 cells. Fixed images of dividing C2C12, Vero and Ptk2 cells in early cytokinesis (C2C12), mid cytokinesis (Vero) and late cytokinesis (Ptk2). Cells were co-stained for mitochondria (MitoTracker Red, red), actin (Phallodin-488, green) and DNA (DAPI, blue). Bar, 10 µm.

    Techniques Used: Staining

    19) Product Images from "Establishment of Baculovirus-Expressed VLPs Induced Syncytial Formation Assay for Flavivirus Antiviral Screening"

    Article Title: Establishment of Baculovirus-Expressed VLPs Induced Syncytial Formation Assay for Flavivirus Antiviral Screening

    Journal: Viruses

    doi: 10.3390/v10070365

    AMS affected ZIKV entry process in Vero cells. ( A ) AMS cytotoxicity was assessed by the viability of Vero cells treated with AMS. Pre-seeded Vero cells were treated with increasing concentrations of AMS for 24 h, and then subjected to cell viability analysis using CCK-8. ( B , C ) AMS affected ZIKV infection in Vero cells at early stage. Vero cells were treated with indicated concentrations of AMS at different time points prior, during, or after the addition of ZIKV strain SZ-WIV01 (100 pfu) and the inhibition efficiency was measured with plaque assay. The plaques of Vero cells treated with AMS during the addition of ZIKV strain SZ-WIV01 ( B ). Plaques were counted and percentage of plaque reduction was calculated ( C ). ( D – F ) AMS affected ZIKV strain SZ-WIV01 replication in Vero cells in a dose-dependent manner. ZIKV strain SZ-WIV01 was incubated with indicated concentrations of AMS and infected Vero cells at an MOI of 0.1. At 48 h p.i., the intracellular level of ZIKV E protein (green) in Vero cells was detected with immunofluorescence analysis ( D ). The titer of ZIKV in the supernatant was quantified by plaque assay ( E ). The intracellular level of ZIKV RNA was quantified with quantitative PCR ( F ). Bars, 50 μm. Data are means ± SD of triplicate experiments. *** p
    Figure Legend Snippet: AMS affected ZIKV entry process in Vero cells. ( A ) AMS cytotoxicity was assessed by the viability of Vero cells treated with AMS. Pre-seeded Vero cells were treated with increasing concentrations of AMS for 24 h, and then subjected to cell viability analysis using CCK-8. ( B , C ) AMS affected ZIKV infection in Vero cells at early stage. Vero cells were treated with indicated concentrations of AMS at different time points prior, during, or after the addition of ZIKV strain SZ-WIV01 (100 pfu) and the inhibition efficiency was measured with plaque assay. The plaques of Vero cells treated with AMS during the addition of ZIKV strain SZ-WIV01 ( B ). Plaques were counted and percentage of plaque reduction was calculated ( C ). ( D – F ) AMS affected ZIKV strain SZ-WIV01 replication in Vero cells in a dose-dependent manner. ZIKV strain SZ-WIV01 was incubated with indicated concentrations of AMS and infected Vero cells at an MOI of 0.1. At 48 h p.i., the intracellular level of ZIKV E protein (green) in Vero cells was detected with immunofluorescence analysis ( D ). The titer of ZIKV in the supernatant was quantified by plaque assay ( E ). The intracellular level of ZIKV RNA was quantified with quantitative PCR ( F ). Bars, 50 μm. Data are means ± SD of triplicate experiments. *** p

    Techniques Used: Affinity Magnetic Separation, CCK-8 Assay, Infection, Inhibition, Plaque Assay, Incubation, Immunofluorescence, Real-time Polymerase Chain Reaction

    20) Product Images from "Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells"

    Article Title: Dynamics of Actin-Based Movement by Rickettsia rickettsii in Vero Cells

    Journal: Infection and Immunity

    doi:

    Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.
    Figure Legend Snippet: Time-lapse video fluorescence microscopy of Listeria and Rickettsia ABM. Vero cells were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Laser-scanning confocal fluorescence microscopy was conducted with live cells, and images were collected every 21 s. (A) Actin-based movement of Listeria . The average rate of movement was 12 μm/min ( n = 23). (B) Actin-based movement of Rickettsia . The average rate of movement was 4.8 μm/min ( n = 28). Bars, 2.5 μm.

    Techniques Used: Fluorescence, Microscopy, Transfection, Plasmid Preparation, Infection

    Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.
    Figure Legend Snippet: Incorporation of GFP-actin into COS-7 cell stress fibers and actin tails of R. rickettsii and Listeria monocytogenes within Vero cells. Mammalian cell cultures were transfected with the plasmid pEGFP-C1/actin encoding GFP–β-actin and infected with either L. monocytogenes or R. rickettsii . Transfected COS-7 cells were fixed and viewed without subsequent staining. Infected Vero cells were fixed and permeabilized, and intracellular bacteria were stained by indirect immunofluorescence with the second antibody conjugated to rhodamine (red). (A) Incorporation of GFP-actin (green) into stress fibers of COS-7 cells. (B) Incorporation of GFP-actin into R. rickettsii actin tails. Note the cluster of organisms undergoing binary fission with one large polar actin tail. (C) Incorporation of GFP-actin into L. monocytogenes actin tails. Bars: panel A, 15 μm; panel B, 2 μm; panel C, 3 μm.

    Techniques Used: Transfection, Plasmid Preparation, Infection, Staining, Immunofluorescence

    21) Product Images from "Newcastle Disease Virus Expressing Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces Strong Mucosal and Serum Antibody Responses in Guinea Pigs ▿"

    Article Title: Newcastle Disease Virus Expressing Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Induces Strong Mucosal and Serum Antibody Responses in Guinea Pigs ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.05050-11

    Oligomeric status of HIV-1 gp160 expressed by rNDVs. Lysates of rLasota- or rLaSota/gp160-infected DF1 (A) and Vero (B) cells, or gradient-purified viruses (C), were cross-linked with DSP at a final concentration of 1 mM. After the cross-linking, the
    Figure Legend Snippet: Oligomeric status of HIV-1 gp160 expressed by rNDVs. Lysates of rLasota- or rLaSota/gp160-infected DF1 (A) and Vero (B) cells, or gradient-purified viruses (C), were cross-linked with DSP at a final concentration of 1 mM. After the cross-linking, the

    Techniques Used: Infection, Purification, Concentration Assay

    Western blot analysis of HIV-1 gp160 expressed by rLaSota/ gp160 in DF1 and Vero cells. DF1 (A) and Vero (B) cells were infected with rLaSota or rLaSota/gp160 virus at an MOI of 0.01 PFU. After 48 h, the cells were collected and processed to prepare cell
    Figure Legend Snippet: Western blot analysis of HIV-1 gp160 expressed by rLaSota/ gp160 in DF1 and Vero cells. DF1 (A) and Vero (B) cells were infected with rLaSota or rLaSota/gp160 virus at an MOI of 0.01 PFU. After 48 h, the cells were collected and processed to prepare cell

    Techniques Used: Western Blot, Infection

    Detection of expression of HIV-1 gp160 by immunofluorescence. Vero cells were infected with rLaSota (panels a and c) or rLaSota/gp160 (panels b and d) at an MOI of 0.1 PFU. After 24 h, the infected cells were fixed with paraformaldehyde and permeabilized
    Figure Legend Snippet: Detection of expression of HIV-1 gp160 by immunofluorescence. Vero cells were infected with rLaSota (panels a and c) or rLaSota/gp160 (panels b and d) at an MOI of 0.1 PFU. After 24 h, the infected cells were fixed with paraformaldehyde and permeabilized

    Techniques Used: Expressing, Immunofluorescence, Infection

    22) Product Images from "A single-amino acid substitution in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine, a flavivirus inhibitor"

    Article Title: A single-amino acid substitution in West Nile virus 2K peptide between NS4A and NS4B confers resistance to lycorine, a flavivirus inhibitor

    Journal: Virology

    doi: 10.1016/j.virol.2008.11.003

    Identification of lycorine as an inhibitor of WNV and DENV-1 VLP infections. (A) Structure of lycorine. The carbon positions of the lycorine molecule are numbered. (B) Production of flavivirus VLPs. Flavivirus VLPs were prepared by sequential transfection of BHK-21 cells with a luciferase-reporting replicon (Flavi-Rluc2A-Rep) and an SFV vector expressing flavivirus structural proteins (SFV-Flavi-CprME). See Materials and Methods for details. (C) Inhibition of WNV and DENV-1 VLP infections by lycorine. Vero cells were infected with WNV (1 FFU/cell) or DENV-1 (0.01 FFU/cell) VLPs in the presence of 1.5 μM lycorine. Luciferase activities were measured at 24 h post-infection. Average results of three independent experiments are shown.
    Figure Legend Snippet: Identification of lycorine as an inhibitor of WNV and DENV-1 VLP infections. (A) Structure of lycorine. The carbon positions of the lycorine molecule are numbered. (B) Production of flavivirus VLPs. Flavivirus VLPs were prepared by sequential transfection of BHK-21 cells with a luciferase-reporting replicon (Flavi-Rluc2A-Rep) and an SFV vector expressing flavivirus structural proteins (SFV-Flavi-CprME). See Materials and Methods for details. (C) Inhibition of WNV and DENV-1 VLP infections by lycorine. Vero cells were infected with WNV (1 FFU/cell) or DENV-1 (0.01 FFU/cell) VLPs in the presence of 1.5 μM lycorine. Luciferase activities were measured at 24 h post-infection. Average results of three independent experiments are shown.

    Techniques Used: Transfection, Luciferase, Plasmid Preparation, Expressing, Inhibition, Infection

    23) Product Images from "The Thr205 Phosphorylation Site within Respiratory Syncytial Virus Matrix (M) Protein Modulates M Oligomerization and Virus Production"

    Article Title: The Thr205 Phosphorylation Site within Respiratory Syncytial Virus Matrix (M) Protein Modulates M Oligomerization and Virus Production

    Journal: Journal of Virology

    doi: 10.1128/JVI.03856-13

    CK2 activity enhances RSV infectious virus production. (A) A549 cells were treated for 48 h with 20 nM siRNA specific for CK2α or control siRNA, followed by infection with RSV at an MOI of 1 for 24 h. Cells were harvested, and cell-associated virus was analyzed by plaque assay on Vero cells. Results shown are represented as the percentages of plaque compared to that of the negative control and are the means ± standard errors of the means (SEM) ( n = 3 experiments); significant difference (Student's t test) is indicated by the P ) for 12 h either 6 or 18 h postinfection (p.i.). In the case of Vero cells, cell-associated virus was harvested at 30 h and 48 h p.i. and titrated on Vero cells. In the case of HEp-2 cells, culture supernatants and cell-associated virus were collected at 48 h p.i. and infectious virus titers were determined on Vero cells. Results for log TCID 50 /ml (mean ± SEM, n = 3 experiments) are shown as percentages relative to the control (no TBB). (C) Vero and HEp-2 cells were infected with RSV A2 at an MOI of 1. Cells were treated without or with 2.8 ng/ml TBB for 12 h at 6 to 18 h p.i. or 18 to 30 h p.i. The medium was then replaced with fresh medium without TBB, and cells were cultured further up to 48 h p.i. Cell toxicity was determined by LDH release in culture supernatants as described in Materials and Methods. Data (means ± standard deviations [SD]) from two independent experiments are represented as LDH activity, determined in triplicate, relative to no treatment.
    Figure Legend Snippet: CK2 activity enhances RSV infectious virus production. (A) A549 cells were treated for 48 h with 20 nM siRNA specific for CK2α or control siRNA, followed by infection with RSV at an MOI of 1 for 24 h. Cells were harvested, and cell-associated virus was analyzed by plaque assay on Vero cells. Results shown are represented as the percentages of plaque compared to that of the negative control and are the means ± standard errors of the means (SEM) ( n = 3 experiments); significant difference (Student's t test) is indicated by the P ) for 12 h either 6 or 18 h postinfection (p.i.). In the case of Vero cells, cell-associated virus was harvested at 30 h and 48 h p.i. and titrated on Vero cells. In the case of HEp-2 cells, culture supernatants and cell-associated virus were collected at 48 h p.i. and infectious virus titers were determined on Vero cells. Results for log TCID 50 /ml (mean ± SEM, n = 3 experiments) are shown as percentages relative to the control (no TBB). (C) Vero and HEp-2 cells were infected with RSV A2 at an MOI of 1. Cells were treated without or with 2.8 ng/ml TBB for 12 h at 6 to 18 h p.i. or 18 to 30 h p.i. The medium was then replaced with fresh medium without TBB, and cells were cultured further up to 48 h p.i. Cell toxicity was determined by LDH release in culture supernatants as described in Materials and Methods. Data (means ± standard deviations [SD]) from two independent experiments are represented as LDH activity, determined in triplicate, relative to no treatment.

    Techniques Used: Activity Assay, Infection, Plaque Assay, Negative Control, Cell Culture

    24) Product Images from "Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes"

    Article Title: Conserved motifs in the hypervariable domain of chikungunya virus nsP3 required for transmission by Aedes aegypti mosquitoes

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0006958

    At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.
    Figure Legend Snippet: At least one FGDF motif is required for the interaction of nsP3 with G3BP and mosquito Rin. (A) Schematic overview of the used plasmids expressing Rasputin (Rin) fused to EGFP Rin-EGFP or mCherry Rin-mC and CHIKV nsP3 EGFP fusion proteins of wild type and FGDF single- and double-mutant nsP3. CMV cytomegalovirus promoter; PUB Aedes aegypti poly-ubiquitin promoter; NTF2 nuclear transport factor 2-like domain; RRM RNA recognition motif; RGG arginine glycine rich region; HVD hypervariable domain. (B) Vero cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hours post infection (hpi) cells were fixed, permeabilized, stained with α-G3BP and visualized by fluorescent microscopy. (C) Aag2 cells were transfected with pPUB-Rin-EGFP and at 24 hours post transfection (hpt) cells were infected with CHIK IC nsP3mC, CHIK IC nsP3mC-FG N or CHIK IC nsP3mC-FG C . At 24 hpi cells were fixed and visualized by fluorescence microscopy. (D) Vero cells were transfected with CMV driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG C , nsP3EGFP-FG N or nsP3EGFP-FG NC . At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-G3BP and α-GFP antibodies. (E) Aag2 cells were transfected with PUB driven plasmids expressing EGFP, nsP3EGFP, nsP3EGFP-FG N , nsP3EGFP-FG C or nsP3EGFP-FG NC and co-transfected with pPUB-Rin-mC. At 24 hpt cells were lysed and lysates were subjected to co-immunoprecipitation with α-GFP beads. Lysates and co-precipitates were subjected to western blot with α-GFP and α-mCherry antibodies.

    Techniques Used: Expressing, Mutagenesis, Infection, Staining, Microscopy, Transfection, Fluorescence, Immunoprecipitation, Western Blot

    At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.
    Figure Legend Snippet: At least one FGDF motif is required for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC, CHIK IC nsP3mC-FG N , CHIK IC nsP3mC-FG C , or CHIK IC nsP3mC-FG NC . Cells were fixed at 36 hours post transfection, stained with Hoechst, and fluorescence was observed by fluorescence microscopy. (B) Vero cells were transfected with in vitro transcribed RNA of CHIK IC nsP3mC or CHIK IC nsP3-FG NC either individually or co-transfected with in vitro transcribed RNA of CHIK IC . Cells were fixed at 36 hpt, stained with Hoechst and fluorescence was observed by fluorescence microscopy. (C) Vero and C6/36 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep -FG NC and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±SEM, normalized to the wild type replicon from at least three independent experiments. (D) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Vero cells infected in duplicate with an MOI of 0.01 based on end-point dilution assay (EPDA) on Vero cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Vero cells. (E) Growth curves of CHIKV IC , CHIKV IC -FG N and CHIKV IC -FG C on Aag2 cells infected in duplicate with an MOI of 0.01 based on infectivity on Aag2 cells. At the indicated time-points the TCID 50 /ml was determined by EPDA on Aag2 cells. Statistics were performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). Asterisks indicate significance compared to the wild type virus. The dotted line in panels D-E indicates the EPDA detection limit.

    Techniques Used: Transfection, In Vitro, Staining, Fluorescence, Microscopy, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

    The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.
    Figure Legend Snippet: The P-rich motif is important but not essential for chikungunya virus replication in mammalian and mosquito cells. (A) Vero and Aag2 cells were transfected with in vitro transcribed RNA of CHIK rep or CHIK rep ΔPVA and the relative luciferase expression was quantified at 24 hpt. Bars indicate the mean relative light units (RLU) ±standard error of the mean from three independent experiments. (B/C) Vero (B) and Aag2 (C) cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at a multiplicity of infection (MOI) of 0.01. TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. (D) Aag2 cells were infected in duplicate with CHIK IC , CHIK IC P398A or CHIK IC PPR401AAA at an MOI of 5 and the TCID 50 /ml was determined by EPDA on Vero cells at the indicated time-points. Asterisks indicate significant differences compared to the wild type virus by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data at each time-point (α = 0.05). The dotted line in panels B-D indicates the EPDA detection limit.

    Techniques Used: Transfection, In Vitro, Luciferase, Expressing, Infection, End-point Dilution Assay, Transformation Assay

    A single FGDF motif is sufficient for the transmission of chikungunya virus by Aedes aegypti mosquitoes. (A) Schematic experimental set-up. Female Ae . aegypti mosquitoes were infected through an infectious bloodmeal containing 2.8 × 10 5 TCID 50 /ml of CHIK IC , CHIK IC -FG N , CHIK IC -FG C. At 7 days post infection (7dpi) the infection and transmission rates were determined by infectivity assay on (B) Vero and (C) Aag2 cells. Bars represent cumulative numbers from three independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) Ratio between the number of positive bodies or salivas in Vero compared to Aag2 cells. Statistics were performed by Kruskal-Wallis test with Dunn’s post-hoc test (α = 0.05). (E) TCID 50 /ml in the bodies of Ae . aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Aag2 cells. Statistics was performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data (α = 0.05).
    Figure Legend Snippet: A single FGDF motif is sufficient for the transmission of chikungunya virus by Aedes aegypti mosquitoes. (A) Schematic experimental set-up. Female Ae . aegypti mosquitoes were infected through an infectious bloodmeal containing 2.8 × 10 5 TCID 50 /ml of CHIK IC , CHIK IC -FG N , CHIK IC -FG C. At 7 days post infection (7dpi) the infection and transmission rates were determined by infectivity assay on (B) Vero and (C) Aag2 cells. Bars represent cumulative numbers from three independent experiments. n = total number of mosquitoes used per treatment. Statistics were performed by Fisher’s exact test (α = 0.05). (D) Ratio between the number of positive bodies or salivas in Vero compared to Aag2 cells. Statistics were performed by Kruskal-Wallis test with Dunn’s post-hoc test (α = 0.05). (E) TCID 50 /ml in the bodies of Ae . aegypti mosquitoes with CHIKV-positive saliva at 7 dpi were determined by end-point dilution assay on Aag2 cells. Statistics was performed by one-way ANOVA with Tukey’s post-hoc test on Log 10 transformed data (α = 0.05).

    Techniques Used: Transmission Assay, Infection, End-point Dilution Assay, Transformation Assay

    25) Product Images from "The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain"

    Article Title: The Characterization of Immunoprotection Induced by a cDNA Clone Derived from the Attenuated Taiwan Porcine Epidemic Diarrhea Virus Pintung 52 Strain

    Journal: Viruses

    doi: 10.3390/v10100543

    The in vitro characterization of the recombinant iPEDVPT-P96 virus. ( A ) The cytopathic effect in Vero cells infected with the iPEDVPT-P96 virus ( Aa ), PEDVPT-P96 virus ( Ab ), or mock-treated ( Ac ), and the immunocytochemistry ( Ad ) detecting PEDV N protein. The microphotographs were taken at 400× magnification. ( B ) Sequence analysis indicated the appropriate marker mutations of cysteine to thymidine at nucleotide site 24341 and, thymidine to cysteine at nucleotide site 24841, as indicated by the red boxes. ( C ) Plaque morphologies of Vero cells infected with the iPEDVPT-P96 virus, PEDVPT-P96 virus, or mock-infected, acquired after three days of incubation. Note that the iPEDVPT-P96 infection resulted in more consistent plaque sizes. ( D ) The growth kinetics of the iPEDVPT-P96 and PEDVPT-P96 viruses in the Vero cells after infection at 0.01 MOI. The titers of each virus at the indicated time points (hours post-infection; HPI) were expressed as the mean ± standard deviation. Different alphabetic letters indicated significant differences between the iPEDVPT-P96 or PEDVPT-P96 groups ( p
    Figure Legend Snippet: The in vitro characterization of the recombinant iPEDVPT-P96 virus. ( A ) The cytopathic effect in Vero cells infected with the iPEDVPT-P96 virus ( Aa ), PEDVPT-P96 virus ( Ab ), or mock-treated ( Ac ), and the immunocytochemistry ( Ad ) detecting PEDV N protein. The microphotographs were taken at 400× magnification. ( B ) Sequence analysis indicated the appropriate marker mutations of cysteine to thymidine at nucleotide site 24341 and, thymidine to cysteine at nucleotide site 24841, as indicated by the red boxes. ( C ) Plaque morphologies of Vero cells infected with the iPEDVPT-P96 virus, PEDVPT-P96 virus, or mock-infected, acquired after three days of incubation. Note that the iPEDVPT-P96 infection resulted in more consistent plaque sizes. ( D ) The growth kinetics of the iPEDVPT-P96 and PEDVPT-P96 viruses in the Vero cells after infection at 0.01 MOI. The titers of each virus at the indicated time points (hours post-infection; HPI) were expressed as the mean ± standard deviation. Different alphabetic letters indicated significant differences between the iPEDVPT-P96 or PEDVPT-P96 groups ( p

    Techniques Used: In Vitro, Recombinant, Infection, Immunocytochemistry, Sequencing, Marker, Incubation, Standard Deviation

    26) Product Images from "RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma"

    Article Title: RNA-binding protein AUF1 suppresses miR-122 biogenesis by down-regulating Dicer1 in hepatocellular carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.24079

    AUF1 interacts with the coding region of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-ORF. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-ORF and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) EGFP protein expression in the treated Vero cells. n = 3. ** P
    Figure Legend Snippet: AUF1 interacts with the coding region of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-ORF. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-ORF and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) EGFP protein expression in the treated Vero cells. n = 3. ** P

    Techniques Used: Expressing, Transfection, Fluorescence, Microscopy

    AUF1 interacts with the 3′UTR of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-3′UTR. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-3′UTR and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) The expression of EGFP protein in the treated Vero cells. n = 3. ** P
    Figure Legend Snippet: AUF1 interacts with the 3′UTR of DICER1 mRNA and regulates its expression ( A ) The diagram of pEGFP-Dicer1-3′UTR. ( B ) Vero cells were co-transfected with pEGFP-Dicer1-3′UTR and pmCherry-AUF1 for 24 h, and the expression of EGFP and AUF1 was observed by fluorescence microscopy. ( C ) The fluorescence quantity was measured by fluorospectrometer. ( D ) The expression of EGFP protein in the treated Vero cells. n = 3. ** P

    Techniques Used: Expressing, Transfection, Fluorescence, Microscopy

    27) Product Images from "Vaccine-Induced Skewing of T Cell Responses Protects Against Chikungunya Virus Disease"

    Article Title: Vaccine-Induced Skewing of T Cell Responses Protects Against Chikungunya Virus Disease

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2019.02563

    CHKVf5 vaccines did not protect mice against a footpad CHIKV challenge. (A) Mice were administered MCMV-CHKVf5, MCMV WT, or PBS i.p. for 8 weeks and then analyzed by ELISpot for the presence of T cell responses or challenged. Separate groups of mice were vaccinated with AdV-CHKVf5 or AdV-Control for 2 weeks. (B) Splenocytes from two mice per group were collected and IFNγ ELISpot assays were performed by stimulating the splenocytes with CHIKV peptides incorporated in the CHKVf5 fusion gene. (C) IFNγ ELISpot assay performed using splenocytes from mice vaccinated with the indicated vaccine and challenged with CHIKV SL15649 in the footpad (“#” indicates too numerous to count; “nd” indicates not done). (D) Footpad thickness was measured using calipers from 3 to 16 dpi. (E) At 3 dpi, mice were bled and their serum was titered by limiting dilution plaque assay on confluent monolayers of Vero cells. (F) At 3 dpi, viral RNA extracted from ipsilateral and contralateral ankles and ipsilateral quadriceps was quantified by qRT-PCR. (G) At 4 wpi, viral RNA was extracted from ipsilateral and contralateral ankles and viral RNA levels were measured by qRT-PCR.
    Figure Legend Snippet: CHKVf5 vaccines did not protect mice against a footpad CHIKV challenge. (A) Mice were administered MCMV-CHKVf5, MCMV WT, or PBS i.p. for 8 weeks and then analyzed by ELISpot for the presence of T cell responses or challenged. Separate groups of mice were vaccinated with AdV-CHKVf5 or AdV-Control for 2 weeks. (B) Splenocytes from two mice per group were collected and IFNγ ELISpot assays were performed by stimulating the splenocytes with CHIKV peptides incorporated in the CHKVf5 fusion gene. (C) IFNγ ELISpot assay performed using splenocytes from mice vaccinated with the indicated vaccine and challenged with CHIKV SL15649 in the footpad (“#” indicates too numerous to count; “nd” indicates not done). (D) Footpad thickness was measured using calipers from 3 to 16 dpi. (E) At 3 dpi, mice were bled and their serum was titered by limiting dilution plaque assay on confluent monolayers of Vero cells. (F) At 3 dpi, viral RNA extracted from ipsilateral and contralateral ankles and ipsilateral quadriceps was quantified by qRT-PCR. (G) At 4 wpi, viral RNA was extracted from ipsilateral and contralateral ankles and viral RNA levels were measured by qRT-PCR.

    Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Plaque Assay, Quantitative RT-PCR

    28) Product Images from "Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein"

    Article Title: Endocytosis Plays a Critical Role in Proteolytic Processing of the Hendra Virus Fusion Protein

    Journal:

    doi: 10.1128/JVI.79.20.12643-12649.2005

    Fusion assays. (A) Syncytium assay. Vero cells were cotransfected with empty pCAGGS vector, pCAGGS-wild type F, pCAGGS-Hendra FY525A, or pCAGGS-Hendra F Y525F and pCAGGS-Hendra G. Pictures were taken at 43 h posttransfection using a Nikon Diaphot inverted
    Figure Legend Snippet: Fusion assays. (A) Syncytium assay. Vero cells were cotransfected with empty pCAGGS vector, pCAGGS-wild type F, pCAGGS-Hendra FY525A, or pCAGGS-Hendra F Y525F and pCAGGS-Hendra G. Pictures were taken at 43 h posttransfection using a Nikon Diaphot inverted

    Techniques Used: Plasmid Preparation

    Surface expression of wt and mutant Hendra virus F. Vero cells transfected with pCAGGS-wt or mutant Hendra virus F were metabolically labeled overnight with Tran[ 35 S], biotinylated, and lysed. Lysates were immunoprecipitated, and surface proteins were
    Figure Legend Snippet: Surface expression of wt and mutant Hendra virus F. Vero cells transfected with pCAGGS-wt or mutant Hendra virus F were metabolically labeled overnight with Tran[ 35 S], biotinylated, and lysed. Lysates were immunoprecipitated, and surface proteins were

    Techniques Used: Expressing, Mutagenesis, Transfection, Metabolic Labelling, Labeling, Immunoprecipitation

    Expression of wt and mutant Hendra virus F proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 45 min with Tran[ 35 S], chased for the indicated times, and
    Figure Legend Snippet: Expression of wt and mutant Hendra virus F proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 45 min with Tran[ 35 S], chased for the indicated times, and

    Techniques Used: Expressing, Mutagenesis, Transfection, Metabolic Labelling, Labeling

    Biotinylation of cell surface proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 2 h with Tran[ 35 S] and chased for the indicated times prior to the biotinylation
    Figure Legend Snippet: Biotinylation of cell surface proteins. Vero cells were transfected with pCAGGS-wt Hendra F, pCAGGS-Hendra F Y525A, or pCAGGS-Hendra F Y525F. Cells were metabolically labeled for 2 h with Tran[ 35 S] and chased for the indicated times prior to the biotinylation

    Techniques Used: Transfection, Metabolic Labelling, Labeling

    Endocytosis assay. (A) Vero cells were transfected in duplicate with pCAGGS expressing wild-type or mutant Hendra virus F. Cells were metabolically labeled for 2 h with Tran[ 35 S] and chased for 1 h. Plates were moved to 4°C to inhibit endocytosis,
    Figure Legend Snippet: Endocytosis assay. (A) Vero cells were transfected in duplicate with pCAGGS expressing wild-type or mutant Hendra virus F. Cells were metabolically labeled for 2 h with Tran[ 35 S] and chased for 1 h. Plates were moved to 4°C to inhibit endocytosis,

    Techniques Used: Endocytosis Assay, Transfection, Expressing, Mutagenesis, Metabolic Labelling, Labeling

    29) Product Images from "Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains"

    Article Title: Tet-Inducible Production of Infectious Zika Virus from the Full-Length cDNA Clones of African- and Asian-Lineage Strains

    Journal: Viruses

    doi: 10.3390/v10120700

    Comparison of cDNA-derived MR766 and FSS13025 virus growth. Vero cells (2.5 × 10 5 /well) in 12-well plates were co-transfected with 1 µg pTight-ZIKV/MR766 or pTight-ZIKV/FSS13025 DNA and 1 µg pTet-On vector, followed by the addition of 1 µg/mL Doxycycline to cell culture media. Infectious virus titers in the supernatants collected at day 3 (MR766) or day 6 (FSS13025) were quantified by a plaque assay. ( A ) Plaque formation by cDNA-derived ZIKV/MR766 and ZIKV/FSS13025 viruses. Vero cells in 12-well cell culture plate were infected with 30 PFU of either MR766 or FSS13025. Plaques were stained and visualized after 4 days (for MR766) or 6 days (for FSS13025) p.i. ( B ) Growth curves of cDNA-derived MR766 and FSS13025 viruses. To compare the growth ability between MR766 and FSS13025, 4 × 10 5 Vero cells seeded in 6-well plates were infected with MR766 or FSS13025 virus at 0.01 MOI at 37 °C for 1 h. Upon washing with phosphate-buffered saline (PBS) three times, infected Vero cells were incubated with 2 mL DMEM containing 10% FBS. At day 1, 2, and 3 post-infection, virus in the supernatant was collected and stored at −80 °C. Virus yields at different time points were determined by a plaque assay. Data points represent the mean titer of triplicates. Statistical significance was analyzed by Student’s t -test: ** p
    Figure Legend Snippet: Comparison of cDNA-derived MR766 and FSS13025 virus growth. Vero cells (2.5 × 10 5 /well) in 12-well plates were co-transfected with 1 µg pTight-ZIKV/MR766 or pTight-ZIKV/FSS13025 DNA and 1 µg pTet-On vector, followed by the addition of 1 µg/mL Doxycycline to cell culture media. Infectious virus titers in the supernatants collected at day 3 (MR766) or day 6 (FSS13025) were quantified by a plaque assay. ( A ) Plaque formation by cDNA-derived ZIKV/MR766 and ZIKV/FSS13025 viruses. Vero cells in 12-well cell culture plate were infected with 30 PFU of either MR766 or FSS13025. Plaques were stained and visualized after 4 days (for MR766) or 6 days (for FSS13025) p.i. ( B ) Growth curves of cDNA-derived MR766 and FSS13025 viruses. To compare the growth ability between MR766 and FSS13025, 4 × 10 5 Vero cells seeded in 6-well plates were infected with MR766 or FSS13025 virus at 0.01 MOI at 37 °C for 1 h. Upon washing with phosphate-buffered saline (PBS) three times, infected Vero cells were incubated with 2 mL DMEM containing 10% FBS. At day 1, 2, and 3 post-infection, virus in the supernatant was collected and stored at −80 °C. Virus yields at different time points were determined by a plaque assay. Data points represent the mean titer of triplicates. Statistical significance was analyzed by Student’s t -test: ** p

    Techniques Used: Derivative Assay, Transfection, Plasmid Preparation, Cell Culture, Plaque Assay, Infection, Staining, Incubation

    Production of cDNA-derived infectious ZIKV. ( A ) Production of infectious ZIKV/MR766 virus upon DNA transfection with or without Tet-induction. Vero cells (2.5 × 10 5 /well) in 12-well cell culture plates were transfected with 1 μg of pTight-ZIKV/MR766 DNA and 1 μg of empty vector or pTet-On vector using lipofectamine 2000. At 3 days p.t., the viral E protein in the DNA-transfected cells was detected by immunofluorescence assay (IFA) using an E-specific monoclonal antibody (D1-4G2-4-15). At the same time, the supernatants were used to infect fresh Vero cells. At 3 days p.i., the cytopathic effect (CPE) was recorded and the E protein in the ZIKV/MR766-infected cells was determined by IFA. ( B ) Determination of cDNA-derived ZIKV/FSS13025 replication and production by CPE and IFA. Experiments were carried out in the same way as in ( A ) except that the pTight-ZIKV/FSS13025 DNA was used. At 6 days p.t., the E protein was detected by IFA in the pTight-ZIKV/FSS13025 DNA-transfected Vero cells. The ZIKV/FSS13025 in the supernatant was used to infect Vero cells. At 3 days p.i., CPE was photographed and the E protein was measured by IFA as described in ( A ). Images in ( A , B ) were 200× magnification.
    Figure Legend Snippet: Production of cDNA-derived infectious ZIKV. ( A ) Production of infectious ZIKV/MR766 virus upon DNA transfection with or without Tet-induction. Vero cells (2.5 × 10 5 /well) in 12-well cell culture plates were transfected with 1 μg of pTight-ZIKV/MR766 DNA and 1 μg of empty vector or pTet-On vector using lipofectamine 2000. At 3 days p.t., the viral E protein in the DNA-transfected cells was detected by immunofluorescence assay (IFA) using an E-specific monoclonal antibody (D1-4G2-4-15). At the same time, the supernatants were used to infect fresh Vero cells. At 3 days p.i., the cytopathic effect (CPE) was recorded and the E protein in the ZIKV/MR766-infected cells was determined by IFA. ( B ) Determination of cDNA-derived ZIKV/FSS13025 replication and production by CPE and IFA. Experiments were carried out in the same way as in ( A ) except that the pTight-ZIKV/FSS13025 DNA was used. At 6 days p.t., the E protein was detected by IFA in the pTight-ZIKV/FSS13025 DNA-transfected Vero cells. The ZIKV/FSS13025 in the supernatant was used to infect Vero cells. At 3 days p.i., CPE was photographed and the E protein was measured by IFA as described in ( A ). Images in ( A , B ) were 200× magnification.

    Techniques Used: Derivative Assay, Transfection, Cell Culture, Plasmid Preparation, Immunofluorescence, Infection

    30) Product Images from "220 mutation in the hemagglutinin of avian influenza A (H7N9) virus alters antigenicity during vaccine strain development"

    Article Title: 220 mutation in the hemagglutinin of avian influenza A (H7N9) virus alters antigenicity during vaccine strain development

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.1080/21645515.2017.1419109

    Growth characteristics of different viruses in MDCK cells. V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs; PR8: A/Puerto Rico/8/34 generated by RG. MDCK cell monolayers were infected at a multiplicity of infection of 0.001 of different viruses. The data was the results of three independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0). ** represents p
    Figure Legend Snippet: Growth characteristics of different viruses in MDCK cells. V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs; PR8: A/Puerto Rico/8/34 generated by RG. MDCK cell monolayers were infected at a multiplicity of infection of 0.001 of different viruses. The data was the results of three independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0). ** represents p

    Techniques Used: Generated, Infection, Software

    A Total protein content (left Y axis) and HA proportion (right Y axis). Total protein content per 10 eggs of each virus. HA proportion was determined using the following equation: HA1+HA2 /HA1+HA2+NP+M. B SDS-PAGE profile of various influenza viruses using the same sample amount. M: Marker (KD). PR8: A/Puerto Rico/8/34 generated by RG; V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs. The data was the results of two independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0).** represents p
    Figure Legend Snippet: A Total protein content (left Y axis) and HA proportion (right Y axis). Total protein content per 10 eggs of each virus. HA proportion was determined using the following equation: HA1+HA2 /HA1+HA2+NP+M. B SDS-PAGE profile of various influenza viruses using the same sample amount. M: Marker (KD). PR8: A/Puerto Rico/8/34 generated by RG; V1E1: RG-17SF003V1E1 with initial rescued in Vero cells followed by 1 passage growth in eggs; V1E5: RG-17SF003V1E5 with initial rescued in Vero cells followed by 5 passages growth in eggs. The data was the results of two independent tests and analyzed by two-way ANOVA using GraphPad Prism 5 software package (version 5.0).** represents p

    Techniques Used: SDS Page, Marker, Generated, Software

    31) Product Images from "Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines"

    Article Title: Single Mutation in the Flavivirus Envelope Protein Hinge Region Increases Neurovirulence for Mice and Monkeys but Decreases Viscerotropism for Monkeys: Relevance to Development and Safety Testing of Live, Attenuated Vaccines

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.4.1932-1943.2002

    Independent RNA transfection and passage series of ChimeriVax-JE virus in FRhL and Vero cells, showing emergence of mutations in the prM-E genes by passage level.
    Figure Legend Snippet: Independent RNA transfection and passage series of ChimeriVax-JE virus in FRhL and Vero cells, showing emergence of mutations in the prM-E genes by passage level.

    Techniques Used: Transfection

    32) Product Images from "Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein"

    Article Title: Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    Journal: Virology

    doi: 10.1016/j.virol.2010.05.031

    SARS-CoV S palmitoylation is necessary for cell-cell fusion At 48h post-transfection, Vero cells expressing SARS-CoV S (A) or SARS-CoV S PN (B) were trypsinized then re-plated. At 24h post-trypsinization, the number of nuclei per syncytia was counted; the average of 3 independent experiments ± SEM is shown (C).
    Figure Legend Snippet: SARS-CoV S palmitoylation is necessary for cell-cell fusion At 48h post-transfection, Vero cells expressing SARS-CoV S (A) or SARS-CoV S PN (B) were trypsinized then re-plated. At 24h post-trypsinization, the number of nuclei per syncytia was counted; the average of 3 independent experiments ± SEM is shown (C).

    Techniques Used: Transfection, Expressing

    33) Product Images from "Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells"

    Article Title: Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.00266-16

    Conditioned media from myeloid cells do not transfer infection to HAE. Conditioned media collected from infected immune cells were applied to the apical surface of scratched HAE (A) or Vero-hSLAM cells (B) as indicated. GFP fluorescence was observed 3
    Figure Legend Snippet: Conditioned media from myeloid cells do not transfer infection to HAE. Conditioned media collected from infected immune cells were applied to the apical surface of scratched HAE (A) or Vero-hSLAM cells (B) as indicated. GFP fluorescence was observed 3

    Techniques Used: Infection, Fluorescence

    34) Product Images from "Triggering unfolded protein response by 2-Deoxy-d-glucose inhibits porcine epidemic diarrhea virus propagation"

    Article Title: Triggering unfolded protein response by 2-Deoxy-d-glucose inhibits porcine epidemic diarrhea virus propagation

    Journal: Antiviral Research

    doi: 10.1016/j.antiviral.2014.03.007

    2-DG inhibits PEDV replication and gene expression. (A) Vero cells were treated with 5 mM 2-DG for the indicated time. Cell lysates were analyzed by Western blot using antibody against GRP78. (B) Vero cells were infected with PEDV (MOI = 0.01) after pretreated with 2-DG 24 h for indicated concentrations. Supernatant (virions) and cell lysates were analyzed by Western blot using antibody against PEDV N. (C) 2-DG affected PEDV replication. The cells were incubated either in the presence or absence of 2-DG for 24 h before infected with PEDV (MOI = 1). The cells were harvested for the indicated time for RT-PCR analysis using primers specific for PEDV N gene. The ratio was the gene copy number of N gene in 2-DG treated and mock-treated cells. (D) Vero cells were infected with PEDV (MOI = 0.01) for 48 h after cells treated with various dosages of 2-DG for 24 h. PEDV titers in the supernatants were measured by plaque formation assays. (E, F) 5 × 10 5 Vero cells were infected with PEDV (MOI = 0.01) for 48 h after cells treated with various dosages of 2-DG for 24 h. Total RNA was extracted, and cDNA was synthesized by reverse transcription. The RT-PCR was used to analyze the viral RNA copy numbers in the cells and supernatants (virion).
    Figure Legend Snippet: 2-DG inhibits PEDV replication and gene expression. (A) Vero cells were treated with 5 mM 2-DG for the indicated time. Cell lysates were analyzed by Western blot using antibody against GRP78. (B) Vero cells were infected with PEDV (MOI = 0.01) after pretreated with 2-DG 24 h for indicated concentrations. Supernatant (virions) and cell lysates were analyzed by Western blot using antibody against PEDV N. (C) 2-DG affected PEDV replication. The cells were incubated either in the presence or absence of 2-DG for 24 h before infected with PEDV (MOI = 1). The cells were harvested for the indicated time for RT-PCR analysis using primers specific for PEDV N gene. The ratio was the gene copy number of N gene in 2-DG treated and mock-treated cells. (D) Vero cells were infected with PEDV (MOI = 0.01) for 48 h after cells treated with various dosages of 2-DG for 24 h. PEDV titers in the supernatants were measured by plaque formation assays. (E, F) 5 × 10 5 Vero cells were infected with PEDV (MOI = 0.01) for 48 h after cells treated with various dosages of 2-DG for 24 h. Total RNA was extracted, and cDNA was synthesized by reverse transcription. The RT-PCR was used to analyze the viral RNA copy numbers in the cells and supernatants (virion).

    Techniques Used: Expressing, Western Blot, Infection, Incubation, Reverse Transcription Polymerase Chain Reaction, Synthesized

    2-DG treatment affects virus packaging. (A) Vero cells were pretreated with 10 mM 2-DG for 24 h, and the cells were analyzed by FACS analysis using anti-CD13 polyclonal antibody. (B, C) 2-DG treatment did not affect virus entry as determined by RT-PCR and Western blot analysis. For RT-PCR analysis, the cells were treated with 10 mM 2-DG before virus infection. Bars represent mean percent copy number of viral RNA levels in cells by RT-PCR using primers specific for PEDV N gene. Cell lysates were analyzed by Western blot using antibody against PEDV N. (D) The Vero cells were infected with PEDV for 2 h (MOI = 1), 2-DG (10 mM) was incubated with the cells. The cells were harvested at different time points. Cell lysates were analyzed by Western blot using antibody against PEDV N. (E, F) The Vero cells was treated with different concentrations 2-DG after infected with PEDV (MOI = 0.01) for 2 h, the ratio of PEDV RNA copy numbers between the supernatants and the cell lysates was detected by Q-PCR, and the ratio of the virus titers in supernatants and cell lysates were determined by plaque assay.
    Figure Legend Snippet: 2-DG treatment affects virus packaging. (A) Vero cells were pretreated with 10 mM 2-DG for 24 h, and the cells were analyzed by FACS analysis using anti-CD13 polyclonal antibody. (B, C) 2-DG treatment did not affect virus entry as determined by RT-PCR and Western blot analysis. For RT-PCR analysis, the cells were treated with 10 mM 2-DG before virus infection. Bars represent mean percent copy number of viral RNA levels in cells by RT-PCR using primers specific for PEDV N gene. Cell lysates were analyzed by Western blot using antibody against PEDV N. (D) The Vero cells were infected with PEDV for 2 h (MOI = 1), 2-DG (10 mM) was incubated with the cells. The cells were harvested at different time points. Cell lysates were analyzed by Western blot using antibody against PEDV N. (E, F) The Vero cells was treated with different concentrations 2-DG after infected with PEDV (MOI = 0.01) for 2 h, the ratio of PEDV RNA copy numbers between the supernatants and the cell lysates was detected by Q-PCR, and the ratio of the virus titers in supernatants and cell lysates were determined by plaque assay.

    Techniques Used: FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot, Infection, Incubation, Polymerase Chain Reaction, Plaque Assay

    The impairment of UPR increased PEDV replication. (A) Vero cells were transfected with shRNAs to knockdown protein PERK for 48 h, and then infected with PEDV for 36 h. Scramble shRNA plasmid was used as the negative control. PERK, eIF2α and phos-eIF2α expression and the N protein of PEDV were analyzed by Western blot. β-Actin was used as sample loading control. (B) PEDV titers in the supernatants were measured by plaque formation assays. (C) The RNA level of gene ORF3 from the infected cells was evaluated by Q-PCR. (D, E) Vero cells were infected with PEDV for 2 h, then treated with 4-PBA for 24 h. The Western blot was performed to test eIF2α and phospho-eIF2α expression and the change of N protein of PEDV. β-Actin was used as sample loading control.
    Figure Legend Snippet: The impairment of UPR increased PEDV replication. (A) Vero cells were transfected with shRNAs to knockdown protein PERK for 48 h, and then infected with PEDV for 36 h. Scramble shRNA plasmid was used as the negative control. PERK, eIF2α and phos-eIF2α expression and the N protein of PEDV were analyzed by Western blot. β-Actin was used as sample loading control. (B) PEDV titers in the supernatants were measured by plaque formation assays. (C) The RNA level of gene ORF3 from the infected cells was evaluated by Q-PCR. (D, E) Vero cells were infected with PEDV for 2 h, then treated with 4-PBA for 24 h. The Western blot was performed to test eIF2α and phospho-eIF2α expression and the change of N protein of PEDV. β-Actin was used as sample loading control.

    Techniques Used: Transfection, Infection, shRNA, Plasmid Preparation, Negative Control, Expressing, Western Blot, Polymerase Chain Reaction

    PEDV induces the host cellular UPR signaling pathways. Vero cells were infected with PEDV at MOI of 0.01 or treated with Tu, the samples were collected at 0, 6, 12, 24, 36 and 48 h.p.i. (A–C) The expression of GRP78, PERK and eIF2α in the samples was analyzed by Western blot. β-Actin was used as sample loading control. (D) The cleavage of ATF6 during UPR was analyzed by Western blot. (E) Unspliced (uXBP-1) and spliced (sXBP-1) mRNA were analyzed by RT-PCR using specific primer pairs.
    Figure Legend Snippet: PEDV induces the host cellular UPR signaling pathways. Vero cells were infected with PEDV at MOI of 0.01 or treated with Tu, the samples were collected at 0, 6, 12, 24, 36 and 48 h.p.i. (A–C) The expression of GRP78, PERK and eIF2α in the samples was analyzed by Western blot. β-Actin was used as sample loading control. (D) The cleavage of ATF6 during UPR was analyzed by Western blot. (E) Unspliced (uXBP-1) and spliced (sXBP-1) mRNA were analyzed by RT-PCR using specific primer pairs.

    Techniques Used: Infection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

    35) Product Images from "MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA"

    Article Title: MERS coronavirus nsp1 participates in an efficient propagation through a specific interaction with viral RNA

    Journal: Virology

    doi: 10.1016/j.virol.2017.08.026

    Rigorous viral RNA recognition by nsp1 in viral replication. (A) The predicted SL5C in MERS-CoV structures of the wt (left) and A9G/R13A mutant (right). Introduced nucleotide mutations are shown in a gray box with white letters. (B) Partial RNA structures of the wt (left) and A9G/R13A mutant (right) of MERS-CoV from the region in the black square in Fig. 5 D. Free energies (dG) are shown below. (C) Lysates of 293T cells transfected with either pCAG-CAT, pCAG-nsp1 or pCAG-nsp1-A9G/R13A together pcD-5′-fLuc were immunoprecipitated with anti-FLAG antibody at 24 h posttransfection. RNAs extracted from the precipitates were subjected to Northern blot analysis using a riboprobe for the luciferase gene. (D) Luciferase activities in 293T cells transfected with either pcD-fluc or pcD-5′-fluc together with the indicated expression plasmids were determined at 24 h posttransfection after standardization with those in cells expressing CAT. The values represent the means ±SD from three independent experiments. (E, F) Vero cells were infected with each recombinant MERS-CoV at an MOI of 0.001, and the virus titers of culture supernatants (E) and levels of subgenomic N mRNA in infected cells (F) were determined at 12, 24 and 48 h postinfection. Because the values of mock samples were under LOD, the data of mock were not shown. (G, H) Huh7 cells were infected with each recombinant MERS-CoV at an MOI of 0.001, and the virus titers of culture supernatants (G) and levels of subgenomic N mRNA in infected cells (H) were determined at 6, 12 and 24 h postinfection. Because the values of mock samples were under LOD, the data of mock were not shown. (I) Plaque morphology of the wt and nsp1-A9G/R13A mutant MERS-CoVs following agarose overlay plaque assay using Vero cell. Infected cells were fixed with phosphate-buffered formaldehyde at 3 days postinfection and stained using crystal violet.
    Figure Legend Snippet: Rigorous viral RNA recognition by nsp1 in viral replication. (A) The predicted SL5C in MERS-CoV structures of the wt (left) and A9G/R13A mutant (right). Introduced nucleotide mutations are shown in a gray box with white letters. (B) Partial RNA structures of the wt (left) and A9G/R13A mutant (right) of MERS-CoV from the region in the black square in Fig. 5 D. Free energies (dG) are shown below. (C) Lysates of 293T cells transfected with either pCAG-CAT, pCAG-nsp1 or pCAG-nsp1-A9G/R13A together pcD-5′-fLuc were immunoprecipitated with anti-FLAG antibody at 24 h posttransfection. RNAs extracted from the precipitates were subjected to Northern blot analysis using a riboprobe for the luciferase gene. (D) Luciferase activities in 293T cells transfected with either pcD-fluc or pcD-5′-fluc together with the indicated expression plasmids were determined at 24 h posttransfection after standardization with those in cells expressing CAT. The values represent the means ±SD from three independent experiments. (E, F) Vero cells were infected with each recombinant MERS-CoV at an MOI of 0.001, and the virus titers of culture supernatants (E) and levels of subgenomic N mRNA in infected cells (F) were determined at 12, 24 and 48 h postinfection. Because the values of mock samples were under LOD, the data of mock were not shown. (G, H) Huh7 cells were infected with each recombinant MERS-CoV at an MOI of 0.001, and the virus titers of culture supernatants (G) and levels of subgenomic N mRNA in infected cells (H) were determined at 6, 12 and 24 h postinfection. Because the values of mock samples were under LOD, the data of mock were not shown. (I) Plaque morphology of the wt and nsp1-A9G/R13A mutant MERS-CoVs following agarose overlay plaque assay using Vero cell. Infected cells were fixed with phosphate-buffered formaldehyde at 3 days postinfection and stained using crystal violet.

    Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, Northern Blot, Luciferase, Expressing, Infection, Recombinant, Plaque Assay, Staining

    36) Product Images from "Blood meal acquisition enhances arbovirus replication in mosquitoes through activation of the GABAergic system"

    Article Title: Blood meal acquisition enhances arbovirus replication in mosquitoes through activation of the GABAergic system

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01244-6

    Glutamate-mediated GABA induction by blood ingestion. a – c Feeding blood enhanced the infection of DENV-2 in A. aegypti a , JEV in C. pipiens pallens b and TAHV in C. pipiens pallens c . Either human blood or 1% sucrose (500 µl) was premixed with supernatant from 6 × 10 5 p.f.u./ml of DENV-2-infected a , 5 × 10 5 p.f.u./ml of JEV-infected b or 5 × 10 6 p.f.u./ml of TAHV-infected c Vero cells (cultured in serum-free medium) (500 µl) to feed mosquitoes via an in vitro membrane blood meal. Mosquito infectivity was determined by TaqMan qPCR at 8 days post-blood meal. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents one mosquito. The data upper mosquito number are represented as the percentage of mosquito infection. Differences in the mosquito infective ratios were compared using Fisher’s exact test. d and e Protein ingestion augmented the amount of glutamic acid d and GABA e in the mosquitoes over time after a meal. Either whole human blood or 70 mg/ml BSA was used for oral feeding of A. aegypti . Mosquitoes fed 1% sucrose served as negative controls. The fed mosquitoes were collected over time after feeding and were used to assess the amounts of glutamic acid and GABA present. The numbers on the Y -axis represents the amount of GABA inoculated per mosquito. The data are presented as the mean ± S.E.M. Differences were considered significant if P
    Figure Legend Snippet: Glutamate-mediated GABA induction by blood ingestion. a – c Feeding blood enhanced the infection of DENV-2 in A. aegypti a , JEV in C. pipiens pallens b and TAHV in C. pipiens pallens c . Either human blood or 1% sucrose (500 µl) was premixed with supernatant from 6 × 10 5 p.f.u./ml of DENV-2-infected a , 5 × 10 5 p.f.u./ml of JEV-infected b or 5 × 10 6 p.f.u./ml of TAHV-infected c Vero cells (cultured in serum-free medium) (500 µl) to feed mosquitoes via an in vitro membrane blood meal. Mosquito infectivity was determined by TaqMan qPCR at 8 days post-blood meal. The number of infected mosquitoes relative to the total number of mosquitoes is shown at the top of each column. Each dot represents one mosquito. The data upper mosquito number are represented as the percentage of mosquito infection. Differences in the mosquito infective ratios were compared using Fisher’s exact test. d and e Protein ingestion augmented the amount of glutamic acid d and GABA e in the mosquitoes over time after a meal. Either whole human blood or 70 mg/ml BSA was used for oral feeding of A. aegypti . Mosquitoes fed 1% sucrose served as negative controls. The fed mosquitoes were collected over time after feeding and were used to assess the amounts of glutamic acid and GABA present. The numbers on the Y -axis represents the amount of GABA inoculated per mosquito. The data are presented as the mean ± S.E.M. Differences were considered significant if P

    Techniques Used: Infection, Cell Culture, In Vitro, Real-time Polymerase Chain Reaction, IF-P

    Glutamate-mediated GABA generation facilitates infection of mosquito vectors by mosquito-borne viruses. a The oral introduction of glutamic acid enhanced the prevalence of DENV-2 infection in A. aegypti . A mixture, which contained 1% sucrose (500 µl), supernatant from 6 × 10 5 p.f.u./ml of DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 10 µg/ml or 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. Mosquitoes fed this mixture without glutamic acid served as a negative control. b , c Knockdown of the AaGAD1 gene suppressed glutamate-mediated GABA generation b , resulting in reduced DENV infection c with sucrose feeding. AaGAD1 was silenced by inoculation with AaGAD1 dsRNA. Mosquitoes inoculated with GFP dsRNA served as negative controls. Three days post dsRNA treatment, a mixture that containing 1% sucrose (500 µl), supernatant from DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. d – f Sucrose feeding with glutamic acid enhanced the prevalence of SINV infection in A. aegypti d , JEV infection in C. pipiens pallens e and TAHV infection in C. pipiens pallens f . A mixture, which contained 1% sucrose (500 µl), supernatant from either 2 × 10 6 p.f.u. ml −1 of SINV-infected d , 5 × 10 5 p.f.u./ml of JEV-infected e and 5 × 10 6 p.f.u./ml of TAHV-infected f Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed mosquitoes via an in vitro blood feeding system. Mosquitoes fed on this mixture without glutamic acid served as negative controls. b The numbers on the Y -axis represents the amount of GABA inoculated per mosquito. The results were reproduced twice. a , c – f Mosquito infectivity was determined by TaqMan qPCR or SYBR Green at 8 days post-blood meal. The number of infected mosquitoes relative to the total numbers of mosquitoes is shown at the top of each column. Each dot represents one mosquito. The data upper mosquito number are represented as the percentage of mosquito infection. Differences in the mosquito infective ratios were compared using Fisher’s exact test. a – f The data from at least two independent experiments were combined
    Figure Legend Snippet: Glutamate-mediated GABA generation facilitates infection of mosquito vectors by mosquito-borne viruses. a The oral introduction of glutamic acid enhanced the prevalence of DENV-2 infection in A. aegypti . A mixture, which contained 1% sucrose (500 µl), supernatant from 6 × 10 5 p.f.u./ml of DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 10 µg/ml or 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. Mosquitoes fed this mixture without glutamic acid served as a negative control. b , c Knockdown of the AaGAD1 gene suppressed glutamate-mediated GABA generation b , resulting in reduced DENV infection c with sucrose feeding. AaGAD1 was silenced by inoculation with AaGAD1 dsRNA. Mosquitoes inoculated with GFP dsRNA served as negative controls. Three days post dsRNA treatment, a mixture that containing 1% sucrose (500 µl), supernatant from DENV-2-infected Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed A. aegypti via an in vitro blood feeding system. d – f Sucrose feeding with glutamic acid enhanced the prevalence of SINV infection in A. aegypti d , JEV infection in C. pipiens pallens e and TAHV infection in C. pipiens pallens f . A mixture, which contained 1% sucrose (500 µl), supernatant from either 2 × 10 6 p.f.u. ml −1 of SINV-infected d , 5 × 10 5 p.f.u./ml of JEV-infected e and 5 × 10 6 p.f.u./ml of TAHV-infected f Vero cells (cultured in serum-free medium) (500 µl), and 100 µg/ml glutamic acid, was used to feed mosquitoes via an in vitro blood feeding system. Mosquitoes fed on this mixture without glutamic acid served as negative controls. b The numbers on the Y -axis represents the amount of GABA inoculated per mosquito. The results were reproduced twice. a , c – f Mosquito infectivity was determined by TaqMan qPCR or SYBR Green at 8 days post-blood meal. The number of infected mosquitoes relative to the total numbers of mosquitoes is shown at the top of each column. Each dot represents one mosquito. The data upper mosquito number are represented as the percentage of mosquito infection. Differences in the mosquito infective ratios were compared using Fisher’s exact test. a – f The data from at least two independent experiments were combined

    Techniques Used: Infection, Cell Culture, In Vitro, Negative Control, Real-time Polymerase Chain Reaction, SYBR Green Assay

    37) Product Images from "Axotomy Induces Phasic Alterations in Luman/CREB3 Expression and Nuclear Localization in Injured and Contralateral Uninjured Sensory Neurons: Correlation With Intrinsic Axon Growth Capacity"

    Article Title: Axotomy Induces Phasic Alterations in Luman/CREB3 Expression and Nuclear Localization in Injured and Contralateral Uninjured Sensory Neurons: Correlation With Intrinsic Axon Growth Capacity

    Journal: Journal of Neuropathology and Experimental Neurology

    doi: 10.1093/jnen/nlz008

    Luman antibody and injury specificity controls. (A) Photomicrographs of L5 dorsal root ganglion (DRG) (6-μm sections) processed for immunofluorescence detect Luman protein with anti-Luman rabbit serum (left) and anti-Luman absorbed with cell protein isolates from Luman-transfected Vero cells. Scale bar = 160 μm. Note: Absorption of anti-Luman abolishes immunofluorescence staining. (B) Western blot analysis of anti-Luman rabbit serum-treated membrane of electrophoresed protein extracts from Vero cells transfected with Luman (lane 2) or Zhangfei (lane 3). Molecular weight marker (lane 1). Note: In the Luman-transfected cell extract, anti-Luman recognizes a single band of approximately 60 kDa, the suspected molecular weight of unprocessed Luman, while unable to detect any identifiable antigen in the Zhangfei-transfected cell extract at its expected molecular weight of approximately 30 kDa. (C) Western blot analysis of protein extracts from naïve L4 and L5 DRG (lane 2). Molecular weight marker (lane 1) Note: Anti-Luman recognizes unprocessed full-length Luman (Fl) at approximately 60 kDa and 2 additional faint bands of approximately 40 and 15 kDa, the predicted molecular weights of Luman protein cleavage products (Cl). (D) L5 DRG sections (6 μm) processed to detect injury-associated ATF-3 protein in DRG sections ipsilateral to 1-hour, 1-day, 2-day, 4-day, and 1-week L4–L6 spinal nerve transection or naïve controls. Scale bar = 150 μm. Injury state is confirmed by presence of nuclear ATF-3 immunofluorescence, detectable by 1-day post-injury. (E) To accurately delineate cytoplasmic and nuclear compartments, all tissue was dually processed for Luman (red) and LaminB (green) immunofluorescence, the latter recognizing the nuclear envelope. Only neurons with a clearly defined LaminB ring were analyzed (examples- * ), as they identify cells sectioned through their center with clear cytoplasmic and nuclear compartments; those without defined rings were not (examples- x ). Scale bar = 75 μm.
    Figure Legend Snippet: Luman antibody and injury specificity controls. (A) Photomicrographs of L5 dorsal root ganglion (DRG) (6-μm sections) processed for immunofluorescence detect Luman protein with anti-Luman rabbit serum (left) and anti-Luman absorbed with cell protein isolates from Luman-transfected Vero cells. Scale bar = 160 μm. Note: Absorption of anti-Luman abolishes immunofluorescence staining. (B) Western blot analysis of anti-Luman rabbit serum-treated membrane of electrophoresed protein extracts from Vero cells transfected with Luman (lane 2) or Zhangfei (lane 3). Molecular weight marker (lane 1). Note: In the Luman-transfected cell extract, anti-Luman recognizes a single band of approximately 60 kDa, the suspected molecular weight of unprocessed Luman, while unable to detect any identifiable antigen in the Zhangfei-transfected cell extract at its expected molecular weight of approximately 30 kDa. (C) Western blot analysis of protein extracts from naïve L4 and L5 DRG (lane 2). Molecular weight marker (lane 1) Note: Anti-Luman recognizes unprocessed full-length Luman (Fl) at approximately 60 kDa and 2 additional faint bands of approximately 40 and 15 kDa, the predicted molecular weights of Luman protein cleavage products (Cl). (D) L5 DRG sections (6 μm) processed to detect injury-associated ATF-3 protein in DRG sections ipsilateral to 1-hour, 1-day, 2-day, 4-day, and 1-week L4–L6 spinal nerve transection or naïve controls. Scale bar = 150 μm. Injury state is confirmed by presence of nuclear ATF-3 immunofluorescence, detectable by 1-day post-injury. (E) To accurately delineate cytoplasmic and nuclear compartments, all tissue was dually processed for Luman (red) and LaminB (green) immunofluorescence, the latter recognizing the nuclear envelope. Only neurons with a clearly defined LaminB ring were analyzed (examples- * ), as they identify cells sectioned through their center with clear cytoplasmic and nuclear compartments; those without defined rings were not (examples- x ). Scale bar = 75 μm.

    Techniques Used: Immunofluorescence, Transfection, Staining, Western Blot, Molecular Weight, Marker

    38) Product Images from "Virucidal Activity of Gold Nanoparticles Synthesized by Green Chemistry Using Garlic Extract"

    Article Title: Virucidal Activity of Gold Nanoparticles Synthesized by Green Chemistry Using Garlic Extract

    Journal: Viruses

    doi: 10.3390/v11121111

    Virucidal effects of AuNPs-As. ( A ) Vero cells were infected with MeV previously exposed to AuNPs-As at different times. The virucidal effect was determined by PFU reduction and qPCR assays. PFU count and viral RNA number are given in % to that for the untreated virus-infected control cells (Viral control), which was defined as 100%. ( B ) Representative images of the virucidal effect of AuNPs-As, showing cellular control (CC), viral control (VC), and treatment at different periods of incubation (0, 3, 6 h). The data shown are the mean ± SD from three replicated experiments. α ≤ 0.05 (*) compared vs viral control.
    Figure Legend Snippet: Virucidal effects of AuNPs-As. ( A ) Vero cells were infected with MeV previously exposed to AuNPs-As at different times. The virucidal effect was determined by PFU reduction and qPCR assays. PFU count and viral RNA number are given in % to that for the untreated virus-infected control cells (Viral control), which was defined as 100%. ( B ) Representative images of the virucidal effect of AuNPs-As, showing cellular control (CC), viral control (VC), and treatment at different periods of incubation (0, 3, 6 h). The data shown are the mean ± SD from three replicated experiments. α ≤ 0.05 (*) compared vs viral control.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Incubation

    39) Product Images from "Herpes Simplex Virus 1 Lytic Infection Blocks MicroRNA (miRNA) Biogenesis at the Stage of Nuclear Export of Pre-miRNAs"

    Article Title: Herpes Simplex Virus 1 Lytic Infection Blocks MicroRNA (miRNA) Biogenesis at the Stage of Nuclear Export of Pre-miRNAs

    Journal: mBio

    doi: 10.1128/mBio.02856-18

    Impairment of miRNA biogenesis is dependent on ICP27 and viral DNA synthesis but not on Us11. (A) Us11 expression from WT and mutant viruses. 293T cells were infected with the viruses indicated at the top of the panel (MOI = 5) and harvested at 16 hpi for Western blot analysis. The proteins analyzed are identified to the left of the panel. This experiment was performed three times. (B) Us11 deletion does not affect viral miRNA biogenesis. 293T cells were infected (MOI = 5) with the viruses indicated at the top of the panel. At 16 hpi, cells were harvested and analyzed by Northern blot hybridization for miR-H1 and miR-H2 expression. This experiment was performed several times. (C) Effects of ICP27 deletion on miR-H2 biogenesis. Vero cells were mock infected or infected (MOI = 20) for 24 h with the viruses indicated at the top of the panel. Pre-miR-H2 and miR-H2 expression (top panel) and let-7a expression (bottom) were analyzed by Northern blot hybridization. This experiment was performed twice. (D) Effects of ICP27 deletion on miR-H6 biogenesis in Vero and E11 cells. Vero cells, or E11 cells, which complement ICP27 expression, were infected with the viruses indicated at the top of the panel (MOI = 20) for 24 h, when the cells were analyzed by Northern blot hybridization for miR-H6. Expression of let-7a as a loading control is shown at the bottom. This experiment was performed twice. (E) Relief of the block of nuclear export in the absence of ICP27. 293T cells were mock infected (lane 1) or infected with the viruses indicated at the top of the panel. At 12 hpi, total RNA was isolated from infected cells (T; lanes 1, 2, and 5) or from nuclear (N; lanes 3 and 6) or cytoplasmic (C; lanes 4 and 7) fractions and analyzed as described for Fig. 6 for miR-H6, U1, and lys-tRNA (as indicated to the right of the panel). This experiment was performed twice. (F) Effects of ACV and ActD on miRNA biogenesis. 293T cells were infected (MOI = 5) with WT-BAC (lane 1) as a negative control for miR-138 expression or with WTLyt138 (lanes 2 to 7), as indicated in the top two lines of the panel. Plus signs (+) on the next two lines indicate treatment with ACV at the time of infection (lanes 5 to 7) or with ActD at 8 hpi (lanes 3 and 6), respectively. ACV was added to block viral DNA synthesis, and ActD was added to block RNA synthesis to help assess the stability of pre-miR-138 and mature miR-138. Plus signs on the next two lines indicate whether infected cells were harvested for RNA isolation at 8 hpi (lanes 2 and 5) or 14 hpi (lanes 1, 3, 4, 6, and 7). The image below shows the results of Northern analysis of expression of pre-miR-138-1 and miR-138, as indicated to the left. Pre/mature ratios calculated following band quantification are displayed below the image. let-7a levels as loading controls are shown at the bottom. This experiment was performed twice.
    Figure Legend Snippet: Impairment of miRNA biogenesis is dependent on ICP27 and viral DNA synthesis but not on Us11. (A) Us11 expression from WT and mutant viruses. 293T cells were infected with the viruses indicated at the top of the panel (MOI = 5) and harvested at 16 hpi for Western blot analysis. The proteins analyzed are identified to the left of the panel. This experiment was performed three times. (B) Us11 deletion does not affect viral miRNA biogenesis. 293T cells were infected (MOI = 5) with the viruses indicated at the top of the panel. At 16 hpi, cells were harvested and analyzed by Northern blot hybridization for miR-H1 and miR-H2 expression. This experiment was performed several times. (C) Effects of ICP27 deletion on miR-H2 biogenesis. Vero cells were mock infected or infected (MOI = 20) for 24 h with the viruses indicated at the top of the panel. Pre-miR-H2 and miR-H2 expression (top panel) and let-7a expression (bottom) were analyzed by Northern blot hybridization. This experiment was performed twice. (D) Effects of ICP27 deletion on miR-H6 biogenesis in Vero and E11 cells. Vero cells, or E11 cells, which complement ICP27 expression, were infected with the viruses indicated at the top of the panel (MOI = 20) for 24 h, when the cells were analyzed by Northern blot hybridization for miR-H6. Expression of let-7a as a loading control is shown at the bottom. This experiment was performed twice. (E) Relief of the block of nuclear export in the absence of ICP27. 293T cells were mock infected (lane 1) or infected with the viruses indicated at the top of the panel. At 12 hpi, total RNA was isolated from infected cells (T; lanes 1, 2, and 5) or from nuclear (N; lanes 3 and 6) or cytoplasmic (C; lanes 4 and 7) fractions and analyzed as described for Fig. 6 for miR-H6, U1, and lys-tRNA (as indicated to the right of the panel). This experiment was performed twice. (F) Effects of ACV and ActD on miRNA biogenesis. 293T cells were infected (MOI = 5) with WT-BAC (lane 1) as a negative control for miR-138 expression or with WTLyt138 (lanes 2 to 7), as indicated in the top two lines of the panel. Plus signs (+) on the next two lines indicate treatment with ACV at the time of infection (lanes 5 to 7) or with ActD at 8 hpi (lanes 3 and 6), respectively. ACV was added to block viral DNA synthesis, and ActD was added to block RNA synthesis to help assess the stability of pre-miR-138 and mature miR-138. Plus signs on the next two lines indicate whether infected cells were harvested for RNA isolation at 8 hpi (lanes 2 and 5) or 14 hpi (lanes 1, 3, 4, 6, and 7). The image below shows the results of Northern analysis of expression of pre-miR-138-1 and miR-138, as indicated to the left. Pre/mature ratios calculated following band quantification are displayed below the image. let-7a levels as loading controls are shown at the bottom. This experiment was performed twice.

    Techniques Used: DNA Synthesis, Expressing, Mutagenesis, Infection, Western Blot, Northern Blot, Hybridization, Blocking Assay, Isolation, BAC Assay, Negative Control

    40) Product Images from "Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain"

    Article Title: Measles Virus Fusion Machinery Activated by Sialic Acid Binding Globular Domain

    Journal: Journal of Virology

    doi: 10.1128/JVI.02256-13

    Cis -complementation of the F-triggering activity of fusion-deficient chimeric protein by wt MV H. Cell-to-cell fusion of 293T cells transfected with the indicated receptor binding glycoproteins and MV F with Vero-His cells transfected with the MV receptor CD150 (A) or empty vector (B) was assessed by a β-Gal complementation assay as described in Materials and Methods. Vero-His cells were used as target cells. The values are means (with SD) for results from triplicate samples of a representative experiment repeated at least three times.
    Figure Legend Snippet: Cis -complementation of the F-triggering activity of fusion-deficient chimeric protein by wt MV H. Cell-to-cell fusion of 293T cells transfected with the indicated receptor binding glycoproteins and MV F with Vero-His cells transfected with the MV receptor CD150 (A) or empty vector (B) was assessed by a β-Gal complementation assay as described in Materials and Methods. Vero-His cells were used as target cells. The values are means (with SD) for results from triplicate samples of a representative experiment repeated at least three times.

    Techniques Used: Activity Assay, Transfection, Binding Assay, Plasmid Preparation

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    Article Title: Evolutionary enhancement of Zika virus infectivity in Aedes aegypti mosquitoes
    Article Snippet: .. For ZIKV infection, the Vero cells were maintained in VP-SFM medium (11681-020, Gibco). .. The Vero and 293T cell lines were purchased from the ATCC (CCL-81, CRL-3216).

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    Thermo Fisher hsv infected vero cells
    Western blot analysis to screen for candidate antibody-generating proteins of the live <t>HSV-2</t> 0ΔNLS vaccine. Representative Western blots of (UI) uninfected <t>Vero</t> cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.
    Hsv Infected Vero Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total hsv infected vero cell protein
    Western blot analysis to screen for candidate antibody-generating proteins of the live <t>HSV-2</t> 0ΔNLS vaccine. Representative Western blots of (UI) uninfected <t>Vero</t> cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.
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    Thermo Fisher svv infected vero cells
    ). (C and D) Recombinants C (rSVV.eGFP-2a-ORF9) and D (rSVV.eGFP-ORF66) were generated using BAC mutagenesis. In <t>SVV</t> recombinant C, DNA sequences encoding eGFP were fused to the N terminus of SVV ORF9, with in-frame insertions of sequences encoding peptide 2A from porcine teschovirus 1 between the two ORFs. In SVV recombinant D, DNA sequences encoding eGFP were directly fused to the N terminus of SVV ORF66 sequences. Panels on the right show green fluorescence and immunohistochemical staining (using anti-SVV antiserum) of plaques associated with cytopathic effects in <t>Vero</t> cells and rhesus fibroblasts infected with SVV recombinant C or D.
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    Thermo Fisher vero cell monolayers
    Multistep growth curve of rHPIV3 vectors expressing RSV F. <t>Vero</t> cells were infected in triplicate with an MOI of 0.01 TCID 50 /cell of the indicated viruses. Cells were incubated at <t>32°C</t> and aliquots of culture medium were collected at 24 h intervals over 7 days and replaced by equal volumes of fresh medium. Samples were flash frozen and at a later time virus titers were determined in parallel by limiting dilution on LLC-MK2 cells using an hemadsorption assay, and are reported as log 10 TCID 50 per mL. Mean titers are shown with the standard deviations indicated as vertical error bars. The statistical significance of the differences between the vectors expressing RSV F versus wt rHPIV3 empty backbone on day 3 and 7 post-infection was determined by one-way analysis of variance with Tukey’s multiple-comparisons post-test and are indicated by ** (P
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    Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine. Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine. Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Western Blot, Mass Spectrometry, Incubation, Mouse Assay, Infection

    Two-color Western blot: mouse HSV-2 0ΔNLS antiserum versus rabbit antisera against HSV-2 glycoproteins B, C, and D. Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions were incubated with a 1:20,000 dilution of mouse HSV-2 0ΔNLS antiserum (green signal = mouse IgG) and 1:10,000 dilutions of rabbit antisera specific for (A) HSV-2 gB, (B) HSV-2 gC, or (C) HSV-2 gD (red signal = rabbit IgG).

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Two-color Western blot: mouse HSV-2 0ΔNLS antiserum versus rabbit antisera against HSV-2 glycoproteins B, C, and D. Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions were incubated with a 1:20,000 dilution of mouse HSV-2 0ΔNLS antiserum (green signal = mouse IgG) and 1:10,000 dilutions of rabbit antisera specific for (A) HSV-2 gB, (B) HSV-2 gC, or (C) HSV-2 gD (red signal = rabbit IgG).

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Western Blot, Infection, Purification, Incubation

    Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with (A) gD-2 + alum/MPL adjuvant or (B) HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with (A) gD-2 + alum/MPL adjuvant or (B) HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Western Blot, Binding Assay, Mass Spectrometry, BAC Assay, Incubation, Mouse Assay

    Flow cytometric analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Three-population cytometric analysis comparing IgG antibody-binding to a mixture of uninfected (UI) Vero cells versus Vero cells inoculated with HSV-2 ΔgD-BAC or HSV-2 MS. Each cell population was dispersed, differentially labeled with 0, 0.45, or 6 μM CFSE, fixed, permeabilized, and combined for antibody staining and flow cytometry. (A—D) Mixed populations of test cells were incubated with 1:6,000 dilutions of serum from (A) a naïve mouse or a mouse immunized with (B) gD-2 + alum/MPL, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Pan-HSV-2 IgG binding (y-axes) was detected using APC-labeled goat anti-mouse IgG secondary, and was measured in three gates (dashed columns) at the center of the CFSE-negative, CFSE lo , and CFSE hi populations to compare IgG binding to UI cells, HSV-2 ΔgD + cells, versus HSV-2 MS + cells, respectively. E. Mean ± sem of pan-HSV-2 IgG levels in n = 5 mice per immunization group, as measured by i. the increase in mean fluorescent intensity (ΔMFI) of IgG bound to HSV-2 MS + cells relative to UI cells, and ii. the ΔMFI of IgG bound to ΔgD + cells relative to UI cells.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Flow cytometric analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Three-population cytometric analysis comparing IgG antibody-binding to a mixture of uninfected (UI) Vero cells versus Vero cells inoculated with HSV-2 ΔgD-BAC or HSV-2 MS. Each cell population was dispersed, differentially labeled with 0, 0.45, or 6 μM CFSE, fixed, permeabilized, and combined for antibody staining and flow cytometry. (A—D) Mixed populations of test cells were incubated with 1:6,000 dilutions of serum from (A) a naïve mouse or a mouse immunized with (B) gD-2 + alum/MPL, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Pan-HSV-2 IgG binding (y-axes) was detected using APC-labeled goat anti-mouse IgG secondary, and was measured in three gates (dashed columns) at the center of the CFSE-negative, CFSE lo , and CFSE hi populations to compare IgG binding to UI cells, HSV-2 ΔgD + cells, versus HSV-2 MS + cells, respectively. E. Mean ± sem of pan-HSV-2 IgG levels in n = 5 mice per immunization group, as measured by i. the increase in mean fluorescent intensity (ΔMFI) of IgG bound to HSV-2 MS + cells relative to UI cells, and ii. the ΔMFI of IgG bound to ΔgD + cells relative to UI cells.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Flow Cytometry, Binding Assay, BAC Assay, Mass Spectrometry, Labeling, Staining, Cytometry, Incubation, Infection, Mouse Assay

    Western blot analysis of purified HSV-2 virions segregates candidate HSV-2 0ΔNLS antigens into infected cell proteins versus virion proteins. Representative Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions incubated with 1:20,000 dilutions of serum from (A) a mock-immunized mouse (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of viral proteins in total HSV-2-infected cell samples most commonly targeted by mouse IgG antibodies.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis of purified HSV-2 virions segregates candidate HSV-2 0ΔNLS antigens into infected cell proteins versus virion proteins. Representative Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions incubated with 1:20,000 dilutions of serum from (A) a mock-immunized mouse (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of viral proteins in total HSV-2-infected cell samples most commonly targeted by mouse IgG antibodies.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Western Blot, Purification, Infection, Incubation, Mouse Assay, Mass Spectrometry

    Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum. (A-B) IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. (A) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. (B) Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. (C-D). IP-mass spec experiment #2. (C) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in S3 Fig. (D) Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute > 1% to the total pool of positive HSV-2 peptides (i.e., > 147 peptides); (2) have > 30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., S3 Fig. ); (3) have > 25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum. (A-B) IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. (A) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. (B) Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. (C-D). IP-mass spec experiment #2. (C) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in S3 Fig. (D) Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute > 1% to the total pool of positive HSV-2 peptides (i.e., > 147 peptides); (2) have > 30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., S3 Fig. ); (3) have > 25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Immunoprecipitation, Mass Spectrometry, Infection, Incubation, Staining, Negative Control, Derivative Assay, Sequencing

    Cell lines expressing epitope-tagged HSV-2 antigens: relative abundance of gD-, ICP8-, RR-1-, and VP5-specific antibodies in HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells, cells inoculated with 2.5 pfu/cell of HSV-2 MS, or Vero cell lines that stably express the following, recombinant HSV-2 proteins: gD-FLAG, ICP8-FLAG, RR-1-FLAG, or VP5-FLAG incubated with 1:20,000 dilutions of serum from ( A ) naïve mice or ( C, E ) mice immunized with (C) gD-2 + alum/MPL adjuvant or (E) HSV-2 0ΔNLS. Following incubation with mouse serum, blots were rinsed and re-probed with mouse α-FLAG antibody to validate the relative amount of FLAG-tagged HSV-2 protein on each blot. ( B, D, F ) Normalized amount of IgG antibody bound to recombinant gD, FLAG, RR-1, or VP5 on blots incubated with ( B ) naïve mouse serum (n = 3), ( D ) gD-2 antiserum (n = 3), or ( F ) 0ΔNLS antiserum (n = 6). Levels of bound IgG antibody were normalized to account for blot-to-blot variance in the relative amount of each target based on the relative amount of α-FLAG antibody that bound each recombinant protein. In panel F, ‘**’ denotes that IgG antibody in 0ΔNLS antiserum bound RR-1 to significantly greater levels than gD, ICP8, or VP5 (p

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Cell lines expressing epitope-tagged HSV-2 antigens: relative abundance of gD-, ICP8-, RR-1-, and VP5-specific antibodies in HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells, cells inoculated with 2.5 pfu/cell of HSV-2 MS, or Vero cell lines that stably express the following, recombinant HSV-2 proteins: gD-FLAG, ICP8-FLAG, RR-1-FLAG, or VP5-FLAG incubated with 1:20,000 dilutions of serum from ( A ) naïve mice or ( C, E ) mice immunized with (C) gD-2 + alum/MPL adjuvant or (E) HSV-2 0ΔNLS. Following incubation with mouse serum, blots were rinsed and re-probed with mouse α-FLAG antibody to validate the relative amount of FLAG-tagged HSV-2 protein on each blot. ( B, D, F ) Normalized amount of IgG antibody bound to recombinant gD, FLAG, RR-1, or VP5 on blots incubated with ( B ) naïve mouse serum (n = 3), ( D ) gD-2 antiserum (n = 3), or ( F ) 0ΔNLS antiserum (n = 6). Levels of bound IgG antibody were normalized to account for blot-to-blot variance in the relative amount of each target based on the relative amount of α-FLAG antibody that bound each recombinant protein. In panel F, ‘**’ denotes that IgG antibody in 0ΔNLS antiserum bound RR-1 to significantly greater levels than gD, ICP8, or VP5 (p

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Expressing, Western Blot, Mass Spectrometry, Stable Transfection, Recombinant, Incubation, Mouse Assay

    Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics. ( A and B) Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. ( A ) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). ( B) Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics. ( A and B) Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. ( A ) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). ( B) Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.

    Article Snippet: Western blot analysis Protein lysates from uninfected Vero cells, HSV-infected Vero cells, or sucrose-purified HSV-2 virions were harvested using mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) supplemented with 1M dithiotreitol and Halt protease inhibitor cocktail (Thermo Scientific).

    Techniques: Expressing, Western Blot, Mass Spectrometry, Infection, Labeling, Binding Assay

    Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine. Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis to screen for candidate antibody-generating proteins of the live HSV-2 0ΔNLS vaccine. Representative Western blots of (UI) uninfected Vero cells or cells inoculated with 2.5 pfu/cell of HSV-1 KOS or HSV-2 MS incubated with 1:20,000 dilutions of serum from (A) mock-immunized mice (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS ( ICP0 - ) virus, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of HSV-2 proteins most commonly targeted by mouse IgG antibodies, and the open arrow denotes the MW of gD-2.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Western Blot, Mass Spectrometry, Incubation, Mouse Assay, Infection

    Two-color Western blot: mouse HSV-2 0ΔNLS antiserum versus rabbit antisera against HSV-2 glycoproteins B, C, and D. Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions were incubated with a 1:20,000 dilution of mouse HSV-2 0ΔNLS antiserum (green signal = mouse IgG) and 1:10,000 dilutions of rabbit antisera specific for (A) HSV-2 gB, (B) HSV-2 gC, or (C) HSV-2 gD (red signal = rabbit IgG).

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Two-color Western blot: mouse HSV-2 0ΔNLS antiserum versus rabbit antisera against HSV-2 glycoproteins B, C, and D. Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions were incubated with a 1:20,000 dilution of mouse HSV-2 0ΔNLS antiserum (green signal = mouse IgG) and 1:10,000 dilutions of rabbit antisera specific for (A) HSV-2 gB, (B) HSV-2 gC, or (C) HSV-2 gD (red signal = rabbit IgG).

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Western Blot, Infection, Purification, Incubation

    Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with (A) gD-2 + alum/MPL adjuvant or (B) HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells or cells inoculated with 5 pfu/cell of HSV-1 KOS, a HSV-1 ΔgD virus (KOS-gD6), HSV-2 MS, or a HSV-2 ΔgD virus (HSV-2 ΔgD-BAC) incubated with 1:20,000 dilutions of serum from mice immunized with (A) gD-2 + alum/MPL adjuvant or (B) HSV-2 0ΔNLS. Red diamonds (1–9) denote the positions of viral proteins most commonly targeted by mouse IgG antibodies.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Western Blot, Binding Assay, Mass Spectrometry, BAC Assay, Incubation, Mouse Assay

    Flow cytometric analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Three-population cytometric analysis comparing IgG antibody-binding to a mixture of uninfected (UI) Vero cells versus Vero cells inoculated with HSV-2 ΔgD-BAC or HSV-2 MS. Each cell population was dispersed, differentially labeled with 0, 0.45, or 6 μM CFSE, fixed, permeabilized, and combined for antibody staining and flow cytometry. (A—D) Mixed populations of test cells were incubated with 1:6,000 dilutions of serum from (A) a naïve mouse or a mouse immunized with (B) gD-2 + alum/MPL, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Pan-HSV-2 IgG binding (y-axes) was detected using APC-labeled goat anti-mouse IgG secondary, and was measured in three gates (dashed columns) at the center of the CFSE-negative, CFSE lo , and CFSE hi populations to compare IgG binding to UI cells, HSV-2 ΔgD + cells, versus HSV-2 MS + cells, respectively. E. Mean ± sem of pan-HSV-2 IgG levels in n = 5 mice per immunization group, as measured by i. the increase in mean fluorescent intensity (ΔMFI) of IgG bound to HSV-2 MS + cells relative to UI cells, and ii. the ΔMFI of IgG bound to ΔgD + cells relative to UI cells.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Flow cytometric analysis of HSV gD-antigen-deletion mutants: effect on antibody-binding targets of gD-2 antiserum versus HSV-2 0ΔNLS antiserum. Three-population cytometric analysis comparing IgG antibody-binding to a mixture of uninfected (UI) Vero cells versus Vero cells inoculated with HSV-2 ΔgD-BAC or HSV-2 MS. Each cell population was dispersed, differentially labeled with 0, 0.45, or 6 μM CFSE, fixed, permeabilized, and combined for antibody staining and flow cytometry. (A—D) Mixed populations of test cells were incubated with 1:6,000 dilutions of serum from (A) a naïve mouse or a mouse immunized with (B) gD-2 + alum/MPL, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Pan-HSV-2 IgG binding (y-axes) was detected using APC-labeled goat anti-mouse IgG secondary, and was measured in three gates (dashed columns) at the center of the CFSE-negative, CFSE lo , and CFSE hi populations to compare IgG binding to UI cells, HSV-2 ΔgD + cells, versus HSV-2 MS + cells, respectively. E. Mean ± sem of pan-HSV-2 IgG levels in n = 5 mice per immunization group, as measured by i. the increase in mean fluorescent intensity (ΔMFI) of IgG bound to HSV-2 MS + cells relative to UI cells, and ii. the ΔMFI of IgG bound to ΔgD + cells relative to UI cells.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Flow Cytometry, Binding Assay, BAC Assay, Mass Spectrometry, Labeling, Staining, Cytometry, Incubation, Infection, Mouse Assay

    Western blot analysis of purified HSV-2 virions segregates candidate HSV-2 0ΔNLS antigens into infected cell proteins versus virion proteins. Representative Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions incubated with 1:20,000 dilutions of serum from (A) a mock-immunized mouse (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of viral proteins in total HSV-2-infected cell samples most commonly targeted by mouse IgG antibodies.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Western blot analysis of purified HSV-2 virions segregates candidate HSV-2 0ΔNLS antigens into infected cell proteins versus virion proteins. Representative Western blots of (UI) uninfected Vero cells, total HSV-2-infected cell proteins (MOI = 2.5), or sucrose-gradient-purified HSV-2 virions incubated with 1:20,000 dilutions of serum from (A) a mock-immunized mouse (naïve) or mice immunized with (B) gD-2 + alum/MPL adjuvant, (C) HSV-2 0ΔNLS, or (D) an acyclovir-restrained HSV-2 MS infection (MS+ACV). Red diamonds (1–9) denote the positions of viral proteins in total HSV-2-infected cell samples most commonly targeted by mouse IgG antibodies.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Western Blot, Purification, Infection, Incubation, Mouse Assay, Mass Spectrometry

    Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum. (A-B) IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. (A) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. (B) Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. (C-D). IP-mass spec experiment #2. (C) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in S3 Fig. (D) Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute > 1% to the total pool of positive HSV-2 peptides (i.e., > 147 peptides); (2) have > 30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., S3 Fig. ); (3) have > 25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Immunoprecipitation-mass spectrometry (IP-mass spec) analysis as a tool to screen antibody specificities in HSV-2 0ΔNLS antiserum. (A-B) IP-mass spec experiment #1. Uninfected Vero cell proteins (UI Ag) or HSV-2 MS-infected cell proteins (HSV-2 Ag) were resuspended in a NP40-based buffer containing 150 mM NaCl and were incubated with 2% naïve mouse serum or 2% mouse 0ΔNLS-antiserum for 2 hours followed by overnight incubation with Protein A/G agarose beads. (A) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 Ag + mouse 0ΔNLS antiserum versus three negative-control immunoprecipitation reactions. Black arrows denote three protein species pulled down by 0ΔNLS antiserum that were not present in controls. (B) Identity of proteins excised from the gel (panel A), as determined by MALDI-TOF mass spectrometry. (C-D). IP-mass spec experiment #2. (C) Coomassie-blue stained polyacrylamide gel of immunoprecipitates formed by HSV-2 MS-infected cell proteins (HSV-2 Ag) following incubation with 1% mouse 0ΔNLS-antiserum and Protein A/G agarose beads. The entire lane of the gel was analyzed by MALDI-TOF mass spectrometry after being cut into 18 equivalent sized slices (denoted by boxes 1–18); slice-by-slice mass spectrometry identification results for the five most abundant HSV-2 proteins are shown in S3 Fig. (D) Number of peptide matches per positively identified HSV-2 protein. A total of 14,729 peptides were identified by mass spectrometry as being derived from 19 HSV-2 proteins that met our inclusion criteria, which were that a “positive identification” should (1) contribute > 1% to the total pool of positive HSV-2 peptides (i.e., > 147 peptides); (2) have > 30% of its peptides recovered from 3 consecutive gel slices at the protein’s expected MW (e.g., S3 Fig. ); (3) have > 25% of its protein sequence represented were detected by the mass spectrometer, and should (4) yield 10 or more unique peptides. Seventy-two percent of the positive HSV-2 peptides in immunoprecipitates were derived from the 5 most dominant proteins identified; namely, RR-1, ICP8, VP1–2, VP5, and gB.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Immunoprecipitation, Mass Spectrometry, Infection, Incubation, Staining, Negative Control, Derivative Assay, Sequencing

    Cell lines expressing epitope-tagged HSV-2 antigens: relative abundance of gD-, ICP8-, RR-1-, and VP5-specific antibodies in HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells, cells inoculated with 2.5 pfu/cell of HSV-2 MS, or Vero cell lines that stably express the following, recombinant HSV-2 proteins: gD-FLAG, ICP8-FLAG, RR-1-FLAG, or VP5-FLAG incubated with 1:20,000 dilutions of serum from ( A ) naïve mice or ( C, E ) mice immunized with (C) gD-2 + alum/MPL adjuvant or (E) HSV-2 0ΔNLS. Following incubation with mouse serum, blots were rinsed and re-probed with mouse α-FLAG antibody to validate the relative amount of FLAG-tagged HSV-2 protein on each blot. ( B, D, F ) Normalized amount of IgG antibody bound to recombinant gD, FLAG, RR-1, or VP5 on blots incubated with ( B ) naïve mouse serum (n = 3), ( D ) gD-2 antiserum (n = 3), or ( F ) 0ΔNLS antiserum (n = 6). Levels of bound IgG antibody were normalized to account for blot-to-blot variance in the relative amount of each target based on the relative amount of α-FLAG antibody that bound each recombinant protein. In panel F, ‘**’ denotes that IgG antibody in 0ΔNLS antiserum bound RR-1 to significantly greater levels than gD, ICP8, or VP5 (p

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Cell lines expressing epitope-tagged HSV-2 antigens: relative abundance of gD-, ICP8-, RR-1-, and VP5-specific antibodies in HSV-2 0ΔNLS antiserum. Western blots of (UI) uninfected Vero cells, cells inoculated with 2.5 pfu/cell of HSV-2 MS, or Vero cell lines that stably express the following, recombinant HSV-2 proteins: gD-FLAG, ICP8-FLAG, RR-1-FLAG, or VP5-FLAG incubated with 1:20,000 dilutions of serum from ( A ) naïve mice or ( C, E ) mice immunized with (C) gD-2 + alum/MPL adjuvant or (E) HSV-2 0ΔNLS. Following incubation with mouse serum, blots were rinsed and re-probed with mouse α-FLAG antibody to validate the relative amount of FLAG-tagged HSV-2 protein on each blot. ( B, D, F ) Normalized amount of IgG antibody bound to recombinant gD, FLAG, RR-1, or VP5 on blots incubated with ( B ) naïve mouse serum (n = 3), ( D ) gD-2 antiserum (n = 3), or ( F ) 0ΔNLS antiserum (n = 6). Levels of bound IgG antibody were normalized to account for blot-to-blot variance in the relative amount of each target based on the relative amount of α-FLAG antibody that bound each recombinant protein. In panel F, ‘**’ denotes that IgG antibody in 0ΔNLS antiserum bound RR-1 to significantly greater levels than gD, ICP8, or VP5 (p

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Expressing, Western Blot, Mass Spectrometry, Stable Transfection, Recombinant, Incubation, Mouse Assay

    Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics. ( A and B) Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. ( A ) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). ( B) Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.

    Journal: PLoS ONE

    Article Title: Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

    doi: 10.1371/journal.pone.0116091

    Figure Lengend Snippet: Cycloheximide-release analysis segregates candidate HSV-2 0ΔNLS antigens by IE, E, or L expression kinetics. ( A and B) Western blot of Vero cells that were uninfected (UI) or were inoculated with 5 pfu per cell of HSV-2 0ΔRING or wild-type HSV-2 MS. Virus-infected cells were treated with cycloheximide (CHX) for 10 hours followed by 7 hours of treatment with actinomycin D (ActD; lanes 1 and 6); acyclovir (ACV; lanes 2 and 7); or no drug (VEH; vehicle; lanes 3 and 8). HSV-2 0ΔRING and HSV-2 MS-infected cells that were not drug-treated (lanes 4 and 9) were included as a control, and were harvested at 17 hours p.i. ( A ) Two-color analysis of HSV-2 proteins and GFP-tagged ICP0 (expressed by HSV-2 0ΔRING) labeled with 1:20,000 mouse α-0ΔNLS antiserum (red signal) and 1:5,000 rabbit α-GFP antiserum (green signal). ( B) Grayscale representation of mouse IgG (in 0ΔNLS antiserum) binding to HSV-2 proteins.

    Article Snippet: After heat denaturation, 12 µg of uninfected total Vero cell protein, total HSV-infected Vero cell protein, PageRuler Plus MW markers (Thermo Scientific), and/or 2.5 µg sucrose-purified HSV-2 virions were resolved in an 8% denaturing polyacrylamide gel, and were transferred to nitrocellulose membranes.

    Techniques: Expressing, Western Blot, Mass Spectrometry, Infection, Labeling, Binding Assay

    ). (C and D) Recombinants C (rSVV.eGFP-2a-ORF9) and D (rSVV.eGFP-ORF66) were generated using BAC mutagenesis. In SVV recombinant C, DNA sequences encoding eGFP were fused to the N terminus of SVV ORF9, with in-frame insertions of sequences encoding peptide 2A from porcine teschovirus 1 between the two ORFs. In SVV recombinant D, DNA sequences encoding eGFP were directly fused to the N terminus of SVV ORF66 sequences. Panels on the right show green fluorescence and immunohistochemical staining (using anti-SVV antiserum) of plaques associated with cytopathic effects in Vero cells and rhesus fibroblasts infected with SVV recombinant C or D.

    Journal: Journal of Virology

    Article Title: Attenuation of Simian Varicella Virus Infection by Enhanced Green Fluorescent Protein in Rhesus Macaques

    doi: 10.1128/JVI.02253-17

    Figure Lengend Snippet: ). (C and D) Recombinants C (rSVV.eGFP-2a-ORF9) and D (rSVV.eGFP-ORF66) were generated using BAC mutagenesis. In SVV recombinant C, DNA sequences encoding eGFP were fused to the N terminus of SVV ORF9, with in-frame insertions of sequences encoding peptide 2A from porcine teschovirus 1 between the two ORFs. In SVV recombinant D, DNA sequences encoding eGFP were directly fused to the N terminus of SVV ORF66 sequences. Panels on the right show green fluorescence and immunohistochemical staining (using anti-SVV antiserum) of plaques associated with cytopathic effects in Vero cells and rhesus fibroblasts infected with SVV recombinant C or D.

    Article Snippet: We identified SVV-infected Vero cells using the PrimeFlow RNA assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

    Techniques: Generated, BAC Assay, Mutagenesis, Recombinant, Fluorescence, Immunohistochemistry, Staining, Infection

    Multistep growth curve of rHPIV3 vectors expressing RSV F. Vero cells were infected in triplicate with an MOI of 0.01 TCID 50 /cell of the indicated viruses. Cells were incubated at 32°C and aliquots of culture medium were collected at 24 h intervals over 7 days and replaced by equal volumes of fresh medium. Samples were flash frozen and at a later time virus titers were determined in parallel by limiting dilution on LLC-MK2 cells using an hemadsorption assay, and are reported as log 10 TCID 50 per mL. Mean titers are shown with the standard deviations indicated as vertical error bars. The statistical significance of the differences between the vectors expressing RSV F versus wt rHPIV3 empty backbone on day 3 and 7 post-infection was determined by one-way analysis of variance with Tukey’s multiple-comparisons post-test and are indicated by ** (P

    Journal: PLoS ONE

    Article Title: Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates

    doi: 10.1371/journal.pone.0228572

    Figure Lengend Snippet: Multistep growth curve of rHPIV3 vectors expressing RSV F. Vero cells were infected in triplicate with an MOI of 0.01 TCID 50 /cell of the indicated viruses. Cells were incubated at 32°C and aliquots of culture medium were collected at 24 h intervals over 7 days and replaced by equal volumes of fresh medium. Samples were flash frozen and at a later time virus titers were determined in parallel by limiting dilution on LLC-MK2 cells using an hemadsorption assay, and are reported as log 10 TCID 50 per mL. Mean titers are shown with the standard deviations indicated as vertical error bars. The statistical significance of the differences between the vectors expressing RSV F versus wt rHPIV3 empty backbone on day 3 and 7 post-infection was determined by one-way analysis of variance with Tukey’s multiple-comparisons post-test and are indicated by ** (P

    Article Snippet: After incubation for 30 min at 37°C, the serum/virus mix was transferred onto Vero cell monolayers, rocked for 2 h at 32°C, overlaid with Opti-MEM I containing 2% FBS, 0.8% methylcellulose, 1x L-glutamine, and 50 μg/ml gentamicin (Thermo Fisher Scientific) and incubated for 6 days at 37°C for RSV or 32°C for rHPIV3.

    Techniques: Expressing, Infection, Incubation

    Western blot analysis of infected cell lysates and sucrose gradient-purified virions. (A, B) Analysis of cell-associated proteins. LLC-MK2 (A) or Vero (B) cells were infected with the indicated rHPIV3-RSV-F vectors or wt rHPIV3 at an MOI of 3 TCID 50 /cell or with wt RSV at an MOI of 3 PFU/cell and incubated for 48 h at 32°C, after which cells were lysed in denaturing and reducing sample buffer and analyzed by Western blotting. RSV F was detected with a mouse anti-RSV F MAb; note that expression of F protein by RSV in LLC-MK2 cells (A) was below the level of detection due to inefficient infection by RSV in that cell type. rHPIV3 N and P proteins were detected with rabbit polyclonal hyperimmune serum against HPIV3 virions; rHPIV3 F was detected with a rabbit hyperimmune serum against recombinant purified F ectodomain; and rHPIV3 HN was detected with a hyperimmune rabbit serum raised against an HN peptide. Tubulin was detected on all blots as a loading control using a mouse anti-tubulin MAb. Secondary antibodies are described in Materials and Methods. Representative blots are shown. (C) Analysis of purified virions. LLC-MK2 cells were infected with the indicated rHPIV3-RSV-F vectors or wt rHPIV3 at an MOI of 0.1 TCID 50 /cell, and Vero cells were infected with RSV at an MOI of 0.1 PFU/cell (LLC-MK2 cells were used for rHPIV3, but Vero cells were used for RSV because they are more permissive) and incubated at 32°C. Culture medium supernatants were collected, clarified by low-speed centrifugation, and subjected to sucrose gradient centrifugation to partially purify the virus particles. One μg of total protein from each virus preparation, as measured by BCA assay, was denatured, reduced, and analyzed by Western blotting (as in parts A and B) to quantify packaging of RSV F and rHPIV3 proteins into the vector particles. In panels A-C, blot images are representative of three independent experiments. RSV F protein bands were quantified by densitometry and normalized to tubulin (A and B) or rHPIV3 N protein (C). The values of RSV F from three repeats for each of A, B, and C were plotted in D, E, and F, respectively, as fold change in the amount of RSV F relative to that of the F/preN virus assigned the value of 1.0. Cell-associated RSV F in the LLC-MK2 and Vero cell lysates was predominantly detected as the F 0 precursor and the larger F 1 subunit, respectively, and in virions as the F 1 subunit; these were the forms that were quantified.

    Journal: PLoS ONE

    Article Title: Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates

    doi: 10.1371/journal.pone.0228572

    Figure Lengend Snippet: Western blot analysis of infected cell lysates and sucrose gradient-purified virions. (A, B) Analysis of cell-associated proteins. LLC-MK2 (A) or Vero (B) cells were infected with the indicated rHPIV3-RSV-F vectors or wt rHPIV3 at an MOI of 3 TCID 50 /cell or with wt RSV at an MOI of 3 PFU/cell and incubated for 48 h at 32°C, after which cells were lysed in denaturing and reducing sample buffer and analyzed by Western blotting. RSV F was detected with a mouse anti-RSV F MAb; note that expression of F protein by RSV in LLC-MK2 cells (A) was below the level of detection due to inefficient infection by RSV in that cell type. rHPIV3 N and P proteins were detected with rabbit polyclonal hyperimmune serum against HPIV3 virions; rHPIV3 F was detected with a rabbit hyperimmune serum against recombinant purified F ectodomain; and rHPIV3 HN was detected with a hyperimmune rabbit serum raised against an HN peptide. Tubulin was detected on all blots as a loading control using a mouse anti-tubulin MAb. Secondary antibodies are described in Materials and Methods. Representative blots are shown. (C) Analysis of purified virions. LLC-MK2 cells were infected with the indicated rHPIV3-RSV-F vectors or wt rHPIV3 at an MOI of 0.1 TCID 50 /cell, and Vero cells were infected with RSV at an MOI of 0.1 PFU/cell (LLC-MK2 cells were used for rHPIV3, but Vero cells were used for RSV because they are more permissive) and incubated at 32°C. Culture medium supernatants were collected, clarified by low-speed centrifugation, and subjected to sucrose gradient centrifugation to partially purify the virus particles. One μg of total protein from each virus preparation, as measured by BCA assay, was denatured, reduced, and analyzed by Western blotting (as in parts A and B) to quantify packaging of RSV F and rHPIV3 proteins into the vector particles. In panels A-C, blot images are representative of three independent experiments. RSV F protein bands were quantified by densitometry and normalized to tubulin (A and B) or rHPIV3 N protein (C). The values of RSV F from three repeats for each of A, B, and C were plotted in D, E, and F, respectively, as fold change in the amount of RSV F relative to that of the F/preN virus assigned the value of 1.0. Cell-associated RSV F in the LLC-MK2 and Vero cell lysates was predominantly detected as the F 0 precursor and the larger F 1 subunit, respectively, and in virions as the F 1 subunit; these were the forms that were quantified.

    Article Snippet: After incubation for 30 min at 37°C, the serum/virus mix was transferred onto Vero cell monolayers, rocked for 2 h at 32°C, overlaid with Opti-MEM I containing 2% FBS, 0.8% methylcellulose, 1x L-glutamine, and 50 μg/ml gentamicin (Thermo Fisher Scientific) and incubated for 6 days at 37°C for RSV or 32°C for rHPIV3.

    Techniques: Western Blot, Infection, Purification, Incubation, Expressing, Recombinant, Centrifugation, Gradient Centrifugation, BIA-KA, Plasmid Preparation

    Stability of expression of RSV F protein by rHPIV3-RSV-F vectors evaluated by a double-staining immunofluorescence plaque assay. Vero cells were inoculated with 10-fold serial dilutions of the P4 stock of F-H3TMCT/N-P obtained from terminal dilution from transfection #1 and P2 stocks of F/preN, F-H3TMCT/preN, and F/N-P from transfection #2 (see Results ). Cells were infected in duplicate and incubated for 6 days at 32°C under a 0.8% methylcellulose overlay. The cells were fixed and subjected to double-staining immunofluorescence with a rabbit polyclonal hyperimmune serum raised against HPIV3 virions and a mixture of three conformationally-dependent murine anti-RSV F MAbs [ 22 ]. Secondary antibodies were infrared dye-labeled donkey anti-rabbit 800-CW and donkey anti-mouse 680-LT. The Odyssey infrared imaging system was used to acquire plaque images. The infrared dyes were pseudo-colored to appear green and red for rHPIV3 and RSV F antigens, respectively. The plaques co-expressing rHPIV3 and RSV F antigens appear yellow. Plaques in which expression of RSV F could not be detected by the three conformationally-dependent MAbs appear green. Representative monolayers are shown.

    Journal: PLoS ONE

    Article Title: Human parainfluenza virus type 3 expressing the respiratory syncytial virus pre-fusion F protein modified for virion packaging yields protective intranasal vaccine candidates

    doi: 10.1371/journal.pone.0228572

    Figure Lengend Snippet: Stability of expression of RSV F protein by rHPIV3-RSV-F vectors evaluated by a double-staining immunofluorescence plaque assay. Vero cells were inoculated with 10-fold serial dilutions of the P4 stock of F-H3TMCT/N-P obtained from terminal dilution from transfection #1 and P2 stocks of F/preN, F-H3TMCT/preN, and F/N-P from transfection #2 (see Results ). Cells were infected in duplicate and incubated for 6 days at 32°C under a 0.8% methylcellulose overlay. The cells were fixed and subjected to double-staining immunofluorescence with a rabbit polyclonal hyperimmune serum raised against HPIV3 virions and a mixture of three conformationally-dependent murine anti-RSV F MAbs [ 22 ]. Secondary antibodies were infrared dye-labeled donkey anti-rabbit 800-CW and donkey anti-mouse 680-LT. The Odyssey infrared imaging system was used to acquire plaque images. The infrared dyes were pseudo-colored to appear green and red for rHPIV3 and RSV F antigens, respectively. The plaques co-expressing rHPIV3 and RSV F antigens appear yellow. Plaques in which expression of RSV F could not be detected by the three conformationally-dependent MAbs appear green. Representative monolayers are shown.

    Article Snippet: After incubation for 30 min at 37°C, the serum/virus mix was transferred onto Vero cell monolayers, rocked for 2 h at 32°C, overlaid with Opti-MEM I containing 2% FBS, 0.8% methylcellulose, 1x L-glutamine, and 50 μg/ml gentamicin (Thermo Fisher Scientific) and incubated for 6 days at 37°C for RSV or 32°C for rHPIV3.

    Techniques: Expressing, Double Immunofluorescence Staining, Plaque Assay, Transfection, Infection, Incubation, Labeling, Imaging