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Immunofluorescence staining of protein E in <t>BHK-21</t> cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
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1) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

2) Product Images from "Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses"

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0004163

Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.
Figure Legend Snippet: Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

Techniques Used: Mouse Assay, Infection, Construct, Expressing, Luciferase, Adsorption

3) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

4) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

5) Product Images from "An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein"

Article Title: An infectious recombinant foot-and-mouth disease virus expressing a fluorescent marker protein

Journal: The Journal of General Virology

doi: 10.1099/vir.0.052308-0

GFP and Renilla luciferase (RL) ORF insertions into the FMDV genome are deleted. (a) Sequence analysis of the single GFP-FMDV and two RL-FMDV deletion variants. The remaining amino acids of each insertion, as well as those flanking each deletion are shown [see GFP-deleted FMDV and RL-deleted FMDV (1) and (2)]. The amino acids flanking the undeleted insertions are also shown for reference (see GFP-FMDV and RL-FMDV). (b–f) BHK-21 cells transfected with GFP-FMDV and treated with the viral replication inhibitor guanidine hydrochloride (+ Gnd-HCl) or left untreated (− Gnd-HCl). GFP-FMDV replication was clearly visible in the untreated cells as judged by the expression of GFP (green, c) and the non-structural 3A protein (red, d). In comparison, no obvious replication was observed in the Gnd-HCl-treated cells (f). Nuclei are stained blue (DAPI). (g) Confirmation by Western blot analysis of GFP-FMDV replication in BHK-21 cells. Whole-cell lysates were analysed for GFP, the non-structural FMDV 3A protein (and 3A/B precursors, black bar) and γ-tubulin (loading control). Asterisks indicate non-specific bands.
Figure Legend Snippet: GFP and Renilla luciferase (RL) ORF insertions into the FMDV genome are deleted. (a) Sequence analysis of the single GFP-FMDV and two RL-FMDV deletion variants. The remaining amino acids of each insertion, as well as those flanking each deletion are shown [see GFP-deleted FMDV and RL-deleted FMDV (1) and (2)]. The amino acids flanking the undeleted insertions are also shown for reference (see GFP-FMDV and RL-FMDV). (b–f) BHK-21 cells transfected with GFP-FMDV and treated with the viral replication inhibitor guanidine hydrochloride (+ Gnd-HCl) or left untreated (− Gnd-HCl). GFP-FMDV replication was clearly visible in the untreated cells as judged by the expression of GFP (green, c) and the non-structural 3A protein (red, d). In comparison, no obvious replication was observed in the Gnd-HCl-treated cells (f). Nuclei are stained blue (DAPI). (g) Confirmation by Western blot analysis of GFP-FMDV replication in BHK-21 cells. Whole-cell lysates were analysed for GFP, the non-structural FMDV 3A protein (and 3A/B precursors, black bar) and γ-tubulin (loading control). Asterisks indicate non-specific bands.

Techniques Used: Luciferase, Sequencing, Transfection, Expressing, Staining, Western Blot

6) Product Images from "Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus"

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

Journal: Viruses

doi: 10.3390/v10050226

Effects of mutations in SH3 domain ligands on CHIKV RNA infectivity and CHIKV replication. ( A ) BHK-21 cells were electroporated with 1 μg of in vitro transcribed RNAs of CHIKV (wt or bearing mutation in nsP3 HVD); the infectivity of the transcripts was analysed by ICA. Mutant virus RNA infectivity is presented as a percentage of wild-type virus RNA, mean values together with standard error of three independent experiments are shown; ( B ) Growth curve of wt CHIKV or its mutant variants in BHK-21 cells infected at MOI of 0.1. Mean values of four replicates with standard deviation are shown. * denotes p
Figure Legend Snippet: Effects of mutations in SH3 domain ligands on CHIKV RNA infectivity and CHIKV replication. ( A ) BHK-21 cells were electroporated with 1 μg of in vitro transcribed RNAs of CHIKV (wt or bearing mutation in nsP3 HVD); the infectivity of the transcripts was analysed by ICA. Mutant virus RNA infectivity is presented as a percentage of wild-type virus RNA, mean values together with standard error of three independent experiments are shown; ( B ) Growth curve of wt CHIKV or its mutant variants in BHK-21 cells infected at MOI of 0.1. Mean values of four replicates with standard deviation are shown. * denotes p

Techniques Used: Infection, In Vitro, Mutagenesis, Standard Deviation

7) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

8) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

9) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

10) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

11) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

12) Product Images from "Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production"

Article Title: Maintenance of Dimer Conformation by the Dengue Virus Core Protein α4-α4′ Helix Pair Is Critical for Nucleocapsid Formation and Virus Production

Journal: Journal of Virology

doi: 10.1128/JVI.00940-14

The lipid droplet (LD) association ability of CP suggests the exposure of the α2-α2′ hydrophobic region. CP mini-RNA (5 μg) was electroporated into BHK-21 cells (2 × 10 6 cells) and cultured at 37°C with 5% CO 2 . Approximately 4 × 10 5 cells were seeded on glass coverslips in 12-well plates and were fixed at 8 hpt. Fixed cells were permeated with 0.02% TX-100, followed by probing with mouse monoclonal anti-HA antibody (for CP). After secondary fluorescein Cy5-conjugated goat anti-mouse IgG probing, cells were counterpermeated with 0.1% TX-100 prior to Nile Red staining (for LD). Nuclei were stained with DAPI (blue). Cells were visualized using a Zeiss 510 LSM confocal microscope. Panel 1, colocalization of CP-LD in a single plane; panel 2, stacking of 3D images reconstructed from multiple continuous images from Z sectioning (0.35 μm/section); panel 3, 3D surface derived from 3D image of panel 2; sectioning of CP signal revealed the CP-LD interaction (left) and the original image before undergoing sectioning (right); panels 2 and 3 were generated using the Imaris 7.6.5 software; panel 4, CP staining intensity and volume (voxel) of each cell that contains at least 8 LDs were determined, and cells with a CP density of at least 65 AU were selected to determine the surface area of each LD (%) colocalized with CP using Imaris 7.6.5 software. The average surface area of each LD (%) colocalized with CP and the CP density per cell were indicated.
Figure Legend Snippet: The lipid droplet (LD) association ability of CP suggests the exposure of the α2-α2′ hydrophobic region. CP mini-RNA (5 μg) was electroporated into BHK-21 cells (2 × 10 6 cells) and cultured at 37°C with 5% CO 2 . Approximately 4 × 10 5 cells were seeded on glass coverslips in 12-well plates and were fixed at 8 hpt. Fixed cells were permeated with 0.02% TX-100, followed by probing with mouse monoclonal anti-HA antibody (for CP). After secondary fluorescein Cy5-conjugated goat anti-mouse IgG probing, cells were counterpermeated with 0.1% TX-100 prior to Nile Red staining (for LD). Nuclei were stained with DAPI (blue). Cells were visualized using a Zeiss 510 LSM confocal microscope. Panel 1, colocalization of CP-LD in a single plane; panel 2, stacking of 3D images reconstructed from multiple continuous images from Z sectioning (0.35 μm/section); panel 3, 3D surface derived from 3D image of panel 2; sectioning of CP signal revealed the CP-LD interaction (left) and the original image before undergoing sectioning (right); panels 2 and 3 were generated using the Imaris 7.6.5 software; panel 4, CP staining intensity and volume (voxel) of each cell that contains at least 8 LDs were determined, and cells with a CP density of at least 65 AU were selected to determine the surface area of each LD (%) colocalized with CP using Imaris 7.6.5 software. The average surface area of each LD (%) colocalized with CP and the CP density per cell were indicated.

Techniques Used: Cell Culture, Staining, Microscopy, Derivative Assay, Generated, Software

Characterization of FL RNA in BHK-21 cells. (A) Immunofluorescence detection of intracellular viral components of FL RNA-electroporated cells at 48 h posttransfection. Viral components in infected cells were detected using rabbit polyclonal anti-NS3 antibody, mouse monoclonal dsRNA antibody, mouse monoclonal anti-E antibody, or mouse monoclonal anti-DENV-2 CP antibody. Protein staining was performed with FITC-conjugated goat anti-rabbit antibody for NS3 or Cy5-conjugated goat anti-mouse antibody for dsRNA, E, and CP. (B) Translation of transfected FL RNA. Cell lysate from approximately 2 × 10 5 cells were prepared at 4 hpt for Western detection of viral NS3 protein expression. GAPDH detection served as the loading control. (C) Extracellular and intracellular virus production. Viral titers in CF (extracellular fraction) and cell lysate (intracellular fraction) at 96 h post-FL RNA transfection were determined by focus-forming assay. Viral titers are expressed in FFU/μg of electroporated RNA. Data are a representative result of titer determination in triplicates from one transfection experiment. (D) Plaque morphology (upper panel) and focus morphology (lower panel) of virus-producing FL RNAs. Collected CF at 96 h post-FL RNA transfection was subjected to plaque assay and focus-forming assay. (E) Growth kinetics of FL WT-derived virus and DENV-2 PL046. A monolayer of BHK-21 cells in a 6-well dish was infected with virus at an MOI of 0.1 and cultured for 96 h. Titer of virus in cultural fluid (CF) at the indicated period postinfection was determined by FFA. The viral titer is expressed in FFU/ml of CF. Data were obtained from average results of titer determination in triplicates from two independent infections. (F) Immunofluorescence detection of intracellular viral components at 24 h post-virus infection in BHK-21 cells. Cells were infected with FL WT-derived virus and DENV-2 PL046 at an MOI of 1 for 24 h. Viral components in infected cells were detected by immunofluorescence assay (IFA) as described above. (G) Plaque (upper panel) and focus (lower panel) morphologies of FL WT-derived and DENV-2 PL046 viruses. Collected CF at 96 h post-virus infection was subjected to plaque assay and focus-forming assay.
Figure Legend Snippet: Characterization of FL RNA in BHK-21 cells. (A) Immunofluorescence detection of intracellular viral components of FL RNA-electroporated cells at 48 h posttransfection. Viral components in infected cells were detected using rabbit polyclonal anti-NS3 antibody, mouse monoclonal dsRNA antibody, mouse monoclonal anti-E antibody, or mouse monoclonal anti-DENV-2 CP antibody. Protein staining was performed with FITC-conjugated goat anti-rabbit antibody for NS3 or Cy5-conjugated goat anti-mouse antibody for dsRNA, E, and CP. (B) Translation of transfected FL RNA. Cell lysate from approximately 2 × 10 5 cells were prepared at 4 hpt for Western detection of viral NS3 protein expression. GAPDH detection served as the loading control. (C) Extracellular and intracellular virus production. Viral titers in CF (extracellular fraction) and cell lysate (intracellular fraction) at 96 h post-FL RNA transfection were determined by focus-forming assay. Viral titers are expressed in FFU/μg of electroporated RNA. Data are a representative result of titer determination in triplicates from one transfection experiment. (D) Plaque morphology (upper panel) and focus morphology (lower panel) of virus-producing FL RNAs. Collected CF at 96 h post-FL RNA transfection was subjected to plaque assay and focus-forming assay. (E) Growth kinetics of FL WT-derived virus and DENV-2 PL046. A monolayer of BHK-21 cells in a 6-well dish was infected with virus at an MOI of 0.1 and cultured for 96 h. Titer of virus in cultural fluid (CF) at the indicated period postinfection was determined by FFA. The viral titer is expressed in FFU/ml of CF. Data were obtained from average results of titer determination in triplicates from two independent infections. (F) Immunofluorescence detection of intracellular viral components at 24 h post-virus infection in BHK-21 cells. Cells were infected with FL WT-derived virus and DENV-2 PL046 at an MOI of 1 for 24 h. Viral components in infected cells were detected by immunofluorescence assay (IFA) as described above. (G) Plaque (upper panel) and focus (lower panel) morphologies of FL WT-derived and DENV-2 PL046 viruses. Collected CF at 96 h post-virus infection was subjected to plaque assay and focus-forming assay.

Techniques Used: Immunofluorescence, Infection, Staining, Transfection, Western Blot, Expressing, Focus Forming Assay, Plaque Assay, Derivative Assay, Cell Culture

13) Product Images from "Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization"

Article Title: Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization

Journal: Journal of Virology

doi: 10.1128/JVI.00243-13

Characterization of DEN-4 mutants containing silent mutations in DCS-PK. (A) Demonstration of mutants containing silent mutations in the DCS-PK element. Mutations are indicated in the predicted pseudoknot structure of DCS-PK in boldface. The positions of several nucleotides of DCS-PK in the capsid ORF are indicated in the WT structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B) Replication of vRNA mutants in transfected BHK-21 cells. vRNA levels are expressed as fold changes relative to the RNA copies 4 h posttransfection. (C) Growth curves of viruses in the supernatants of vRNA-transfected BHK-21 cells. Virus titers were normalized to transfection efficiency as determined by qRT-PCR at 4 h after transfection. (D) Decay kinetics of various vRNA mutants, which all contained GDD-to-GVD mutations in NS5RdRp. The error bars indicate the standard deviations in all figures.
Figure Legend Snippet: Characterization of DEN-4 mutants containing silent mutations in DCS-PK. (A) Demonstration of mutants containing silent mutations in the DCS-PK element. Mutations are indicated in the predicted pseudoknot structure of DCS-PK in boldface. The positions of several nucleotides of DCS-PK in the capsid ORF are indicated in the WT structure. Note that the secondary structures of DCS-PK mutants are only shown to provide a clear illustration and do not represent the actual folding of these mutants. (B) Replication of vRNA mutants in transfected BHK-21 cells. vRNA levels are expressed as fold changes relative to the RNA copies 4 h posttransfection. (C) Growth curves of viruses in the supernatants of vRNA-transfected BHK-21 cells. Virus titers were normalized to transfection efficiency as determined by qRT-PCR at 4 h after transfection. (D) Decay kinetics of various vRNA mutants, which all contained GDD-to-GVD mutations in NS5RdRp. The error bars indicate the standard deviations in all figures.

Techniques Used: Transfection, Quantitative RT-PCR

14) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

15) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

16) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

17) Product Images from "Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses"

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0004163

Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.
Figure Legend Snippet: Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

Techniques Used: Mouse Assay, Infection, Construct, Expressing, Luciferase, Adsorption

18) Product Images from "Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses"

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0004163

Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.
Figure Legend Snippet: Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

Techniques Used: Mouse Assay, Infection, Construct, Expressing, Luciferase, Adsorption

19) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

20) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

21) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

22) Product Images from "Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells"

Article Title: Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

Journal: Nucleic Acids Research

doi:

( A ) Schematic representation of the mutated envelope polyprotein precursor fused to a cDNA fragment either containing a signal peptide (mGH) or not (mKAP). Arrowheads indicate cleavage by signal peptidase; arrow indicates site of processing of p62mut in the trans-Golgi network; diagonals represent transmembrane domains. ( B ) Cell surface (left) and total (right) E1 revealed by western analysis 24 h after transfection in BHK-21 cells of CMV-based plasmids expressing p62WT fused either to mKAP (lane 1) or mGH (lane 2) and p62mut fused either to mKAP (lane 3) or mGH (lane 4). Arrowheads indicate the position of the E1 protein; the arrow points to the expected position of the unprocessed mKAP-p62mut putative polyprotein. ( C ) Anti-C immunostaining performed on cells 12 h after transfection with p62mut Sindbis virus replicon containing the mKAP fragment (top left) or the mGH fragment (bottom left). Right panels show anti-C immunostaining of cells infected with culture medium from the corresponding transfected cells.
Figure Legend Snippet: ( A ) Schematic representation of the mutated envelope polyprotein precursor fused to a cDNA fragment either containing a signal peptide (mGH) or not (mKAP). Arrowheads indicate cleavage by signal peptidase; arrow indicates site of processing of p62mut in the trans-Golgi network; diagonals represent transmembrane domains. ( B ) Cell surface (left) and total (right) E1 revealed by western analysis 24 h after transfection in BHK-21 cells of CMV-based plasmids expressing p62WT fused either to mKAP (lane 1) or mGH (lane 2) and p62mut fused either to mKAP (lane 3) or mGH (lane 4). Arrowheads indicate the position of the E1 protein; the arrow points to the expected position of the unprocessed mKAP-p62mut putative polyprotein. ( C ) Anti-C immunostaining performed on cells 12 h after transfection with p62mut Sindbis virus replicon containing the mKAP fragment (top left) or the mGH fragment (bottom left). Right panels show anti-C immunostaining of cells infected with culture medium from the corresponding transfected cells.

Techniques Used: Western Blot, Transfection, Expressing, Immunostaining, Infection

23) Product Images from "A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination"

Article Title: A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

Journal: Virology Journal

doi: 10.1186/s12985-017-0775-8

Antigenic characterization of mIBV and rIBV-wt. a Immunofluorescence analyses of IBV Beaudette, MHV A59 and mIBV #1B3-IIA infected cells. LR7 cells infected with mIBV were fixed and double-immunolabeled with a polyclonal against IBV ( green ) and a polyclonal antibody against MHV (red). IBV Beaudette-infected BHK-21 cells and MHV-infected LR7 cells were taken along for comparison. Nuclei are visualized with DAPI ( blue ). Overlay pictures (Merge) are shown on the right. b Similar to ( a ), except that a monoclonal antibody against IBV S2 was used instead of a polyclonal against IBV, indicating the absence of IBV S2 protein in mIBV infected cells. c Immunohistochemistry of IBV H52 BI and rIBV-wt infected CAM tissues. Ten-day-old embryonated chicken eggs were inoculated with IBV H52 BI (positive control), mIBV-infected and p-IBV transcript electroporated LR7 cells (resulting in generation of rIBV-wt), mIBV infected and p-IBV transcript, but not electroporated, LR7 cells (mIBV + p-IBV mock) or PBS (mock). Formalin-fixed and paraffin-embedded CAM tissues were immunohistochemically stained using a monoclonal antibody against IBV S2. Replication of (r)IBV in the epithelial cells of the CAM is indicated by red cytoplasmic staining, which is absent in eggs inoculated with mIBV-infected non-transfected LR7 cells
Figure Legend Snippet: Antigenic characterization of mIBV and rIBV-wt. a Immunofluorescence analyses of IBV Beaudette, MHV A59 and mIBV #1B3-IIA infected cells. LR7 cells infected with mIBV were fixed and double-immunolabeled with a polyclonal against IBV ( green ) and a polyclonal antibody against MHV (red). IBV Beaudette-infected BHK-21 cells and MHV-infected LR7 cells were taken along for comparison. Nuclei are visualized with DAPI ( blue ). Overlay pictures (Merge) are shown on the right. b Similar to ( a ), except that a monoclonal antibody against IBV S2 was used instead of a polyclonal against IBV, indicating the absence of IBV S2 protein in mIBV infected cells. c Immunohistochemistry of IBV H52 BI and rIBV-wt infected CAM tissues. Ten-day-old embryonated chicken eggs were inoculated with IBV H52 BI (positive control), mIBV-infected and p-IBV transcript electroporated LR7 cells (resulting in generation of rIBV-wt), mIBV infected and p-IBV transcript, but not electroporated, LR7 cells (mIBV + p-IBV mock) or PBS (mock). Formalin-fixed and paraffin-embedded CAM tissues were immunohistochemically stained using a monoclonal antibody against IBV S2. Replication of (r)IBV in the epithelial cells of the CAM is indicated by red cytoplasmic staining, which is absent in eggs inoculated with mIBV-infected non-transfected LR7 cells

Techniques Used: Immunofluorescence, Infection, Immunolabeling, Immunohistochemistry, Chick Chorioallantoic Membrane Assay, Positive Control, Staining, Transfection

24) Product Images from "Exclusion of West Nile Virus Superinfection through RNA Replication ▿"

Article Title: Exclusion of West Nile Virus Superinfection through RNA Replication ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01205-09

Mechanism to overcome superinfection exclusion. (A) Attachment/entry of WT and Env mutant WNVs on BHK-21 cells (see Materials and Methods). Relative amounts of viral RNA are presented; viral RNA derived from BHK-21 cells at 4°C without NaCl treatment is set as 100%. Error bars represent standard deviations. (B) Attachment/entry of VLP on BHK-21 cells. VLPs containing WT or E138K mutant Env were prepared. Equal amounts of VLPs (normalized by RNA levels of VLPs) were used to infect BHK-21 cells. Luciferase activities were measured at 2 h p.i. Relative luciferase activities are shown; the luciferase signal from the WT VLP was set as 100%. An average result from three experiments is presented. (C) Effects of Env and prM mutations on VLP assembly/release. BHK-21 cells were sequentially transfected with Rluc2ARep RNA and SFV vector expressing WT or mutant structural genes. At 48 h after the first transfection, real-time RT-PCR was used to quantify intracellular (left) and extracellular replicon RNAs (right). The RNAs derived from the WT structural VLPs were set as 100%. (D) Transient replicon assay. WT and mutant Rluc2ARep RNAs (10 μg each) were electroporated into BHK-21 cells with or without NeoRep and monitored for luciferase activities. An average from quadruplicate results is shown.
Figure Legend Snippet: Mechanism to overcome superinfection exclusion. (A) Attachment/entry of WT and Env mutant WNVs on BHK-21 cells (see Materials and Methods). Relative amounts of viral RNA are presented; viral RNA derived from BHK-21 cells at 4°C without NaCl treatment is set as 100%. Error bars represent standard deviations. (B) Attachment/entry of VLP on BHK-21 cells. VLPs containing WT or E138K mutant Env were prepared. Equal amounts of VLPs (normalized by RNA levels of VLPs) were used to infect BHK-21 cells. Luciferase activities were measured at 2 h p.i. Relative luciferase activities are shown; the luciferase signal from the WT VLP was set as 100%. An average result from three experiments is presented. (C) Effects of Env and prM mutations on VLP assembly/release. BHK-21 cells were sequentially transfected with Rluc2ARep RNA and SFV vector expressing WT or mutant structural genes. At 48 h after the first transfection, real-time RT-PCR was used to quantify intracellular (left) and extracellular replicon RNAs (right). The RNAs derived from the WT structural VLPs were set as 100%. (D) Transient replicon assay. WT and mutant Rluc2ARep RNAs (10 μg each) were electroporated into BHK-21 cells with or without NeoRep and monitored for luciferase activities. An average from quadruplicate results is shown.

Techniques Used: Mutagenesis, Derivative Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

Comparison of growth kinetics of WT and mutant WNVs. Recombinant WNVs containing various mutations recovered from Sel A (A) and Sel C (B) were prepared using an infectious cDNA clone. BHK-21 and NeoRep BHK-21 cells were infected with WT and recombinant viruses at an MOI of 0.1; viral titers in culture fluids were determined by plaque assay on Vero cells. Average results from two independent experiments are shown. (C) VLP infection. VLPs containing the indicated mutations in structural or nonstructural regions were prepared. The amounts of VLPs were quantified by real-time RT-PCR. BHK-21 cells with and without NeoRep were infected with equal amounts of VLPs and assayed for luciferase activities at 24 h posttransfection. The left panel shows average luciferase activities from three independent experiments. The right panel shows the relative luciferase activities (from the left panel); for each cell line, the luciferase signal generated by the WT VLP is set as 1.
Figure Legend Snippet: Comparison of growth kinetics of WT and mutant WNVs. Recombinant WNVs containing various mutations recovered from Sel A (A) and Sel C (B) were prepared using an infectious cDNA clone. BHK-21 and NeoRep BHK-21 cells were infected with WT and recombinant viruses at an MOI of 0.1; viral titers in culture fluids were determined by plaque assay on Vero cells. Average results from two independent experiments are shown. (C) VLP infection. VLPs containing the indicated mutations in structural or nonstructural regions were prepared. The amounts of VLPs were quantified by real-time RT-PCR. BHK-21 cells with and without NeoRep were infected with equal amounts of VLPs and assayed for luciferase activities at 24 h posttransfection. The left panel shows average luciferase activities from three independent experiments. The right panel shows the relative luciferase activities (from the left panel); for each cell line, the luciferase signal generated by the WT VLP is set as 1.

Techniques Used: Mutagenesis, Recombinant, Infection, Plaque Assay, Quantitative RT-PCR, Luciferase, Generated

Superinfection of WNV NeoRep cells with homologous and heterologous viruses. BHK-21 cells with and without WNV NeoRep were infected with SLEV, YFV, DENV-2, WEEV, and VSV at an MOI of 0.1. Viral titers in culture medium were determined by plaque assay on Vero cells. Error bars indicate standard deviations from triplicate experiments.
Figure Legend Snippet: Superinfection of WNV NeoRep cells with homologous and heterologous viruses. BHK-21 cells with and without WNV NeoRep were infected with SLEV, YFV, DENV-2, WEEV, and VSV at an MOI of 0.1. Viral titers in culture medium were determined by plaque assay on Vero cells. Error bars indicate standard deviations from triplicate experiments.

Techniques Used: Infection, Plaque Assay

Restoration of WNV permissiveness by curing replicon RNA from the NeoRep cells. NeoRep BHK-21 cells were treated with a flavivirus inhibitor at 50 μM. Intracellular replicon RNA was monitored by real-time RT-PCR. Three independent treatments were conducted. (A) Cycle threshold (CT) values were presented from cells treated for 0, 3, 8, and 17 rounds. LOD, limit of detection. (B) The real-time RT-PCR results were confirmed by standard RT-PCR using primers targeting the NS3 or NS5 region. The standard RT-PCR resulted in expected sizes of products of 414 and 651 bp, respectively. (C) Viral growth kinetics on BHK-21, NeoRep BHK-21, and cured NeoRep BHK-21 cells. Cells were infected with WNV at an MOI of 0.1. Viral titers in culture medium were determined by plaque assay on Vero cells.
Figure Legend Snippet: Restoration of WNV permissiveness by curing replicon RNA from the NeoRep cells. NeoRep BHK-21 cells were treated with a flavivirus inhibitor at 50 μM. Intracellular replicon RNA was monitored by real-time RT-PCR. Three independent treatments were conducted. (A) Cycle threshold (CT) values were presented from cells treated for 0, 3, 8, and 17 rounds. LOD, limit of detection. (B) The real-time RT-PCR results were confirmed by standard RT-PCR using primers targeting the NS3 or NS5 region. The standard RT-PCR resulted in expected sizes of products of 414 and 651 bp, respectively. (C) Viral growth kinetics on BHK-21, NeoRep BHK-21, and cured NeoRep BHK-21 cells. Cells were infected with WNV at an MOI of 0.1. Viral titers in culture medium were determined by plaque assay on Vero cells.

Techniques Used: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Infection, Plaque Assay

). (D) Immunofluorescence images. BHK-21 and NeoRep BHK-21 cells were transfected with WNV genome-length RNA and stained with antibody against Env protein at 24 h posttransfection. The same view of phase-contrast and fluorescent (Texas Red) images are shown.
Figure Legend Snippet: ). (D) Immunofluorescence images. BHK-21 and NeoRep BHK-21 cells were transfected with WNV genome-length RNA and stained with antibody against Env protein at 24 h posttransfection. The same view of phase-contrast and fluorescent (Texas Red) images are shown.

Techniques Used: Immunofluorescence, Transfection, Staining

Superinfection exclusion occurs at viral RNA synthesis. (A) Viral attach/entry analysis on BHK-21 and NeoRep BHK-21 cells. Experimental details were described in Materials and Methods. Relative amounts of viral RNA are presented; viral RNA derived from BHK-21 cells at 4°C without NaCl treatment is set as 100%. Error bars indicate standard deviations from triplicate results. (B) Transient replication of WNV Rluc2ARep in BHK-21 cells with and without NeoRep. WNV Rluc2A Rep RNA (10 μg) was electroporated into BHK-21 cells with or without NeoRep. The transfected cells were resuspended in 25 ml of DMEM with 10% FBS; 1 ml of cell suspension was seeded into 12-well plates; luciferase activities were measured at the indicated time points. An average of triplicate results is shown. (C) WNV VLP infection assay. BHK-21 and NeoRep BHK-21 cells (4 × 10 4 per well of 96-well plate) were infected with equal amounts of VLPs (1 FFU/cell) and measured for luciferase activities at the indicated time points. Average results from three independent experiments are represented.
Figure Legend Snippet: Superinfection exclusion occurs at viral RNA synthesis. (A) Viral attach/entry analysis on BHK-21 and NeoRep BHK-21 cells. Experimental details were described in Materials and Methods. Relative amounts of viral RNA are presented; viral RNA derived from BHK-21 cells at 4°C without NaCl treatment is set as 100%. Error bars indicate standard deviations from triplicate results. (B) Transient replication of WNV Rluc2ARep in BHK-21 cells with and without NeoRep. WNV Rluc2A Rep RNA (10 μg) was electroporated into BHK-21 cells with or without NeoRep. The transfected cells were resuspended in 25 ml of DMEM with 10% FBS; 1 ml of cell suspension was seeded into 12-well plates; luciferase activities were measured at the indicated time points. An average of triplicate results is shown. (C) WNV VLP infection assay. BHK-21 and NeoRep BHK-21 cells (4 × 10 4 per well of 96-well plate) were infected with equal amounts of VLPs (1 FFU/cell) and measured for luciferase activities at the indicated time points. Average results from three independent experiments are represented.

Techniques Used: Derivative Assay, Transfection, Luciferase, Infection

Selection and characterization of superinfecting WNV. (A) Scheme for selection of superinfecting WNV. Four independent selections were performed on NeoRep BHK-21 cells. (B) Growth kinetics of the selected WNV variants on NeoRep cells. NeoRep BHK-21 cells were infected with WT or selected variants (from the seventh passage) at an MOI of 0.1. Viral titers in culture fluids were determined by plaque assays on Vero cells. Average data from two independent experiments are presented. (C) Infectious center assay of WNV variants on NeoRep BHK-21 cells. The immunostaining-based infectious center assay was described in Materials and Methods. (D) Summary of mutations identified from the four selected superinfecting WNVs. Locations of nucleotide and amino acid changes are indicated.
Figure Legend Snippet: Selection and characterization of superinfecting WNV. (A) Scheme for selection of superinfecting WNV. Four independent selections were performed on NeoRep BHK-21 cells. (B) Growth kinetics of the selected WNV variants on NeoRep cells. NeoRep BHK-21 cells were infected with WT or selected variants (from the seventh passage) at an MOI of 0.1. Viral titers in culture fluids were determined by plaque assays on Vero cells. Average data from two independent experiments are presented. (C) Infectious center assay of WNV variants on NeoRep BHK-21 cells. The immunostaining-based infectious center assay was described in Materials and Methods. (D) Summary of mutations identified from the four selected superinfecting WNVs. Locations of nucleotide and amino acid changes are indicated.

Techniques Used: Selection, Infection, Immunostaining

25) Product Images from "Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus ▿"

Article Title: Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus ▿

Journal:

doi: 10.1128/JVI.00587-08

Infectivity of cleavage site mutants in cell culture. BHK-21 cells were transfected with wild-type or mutant RNAs as indicated on the left. Chymotrypsin was added to the cell culture medium to a final concentration of 5 μg ml −1 . Supernatants
Figure Legend Snippet: Infectivity of cleavage site mutants in cell culture. BHK-21 cells were transfected with wild-type or mutant RNAs as indicated on the left. Chymotrypsin was added to the cell culture medium to a final concentration of 5 μg ml −1 . Supernatants

Techniques Used: Infection, Cell Culture, Transfection, Mutagenesis, Concentration Assay

Neurovirulence of mutant viruses upon intracranial inoculation of suckling mice. Supernatants of transfected BHK-21 cells were normalized by SDS-ELISA to contain equal amounts of protein E (200 ng/ml). Survival of mice was monitored over a time period
Figure Legend Snippet: Neurovirulence of mutant viruses upon intracranial inoculation of suckling mice. Supernatants of transfected BHK-21 cells were normalized by SDS-ELISA to contain equal amounts of protein E (200 ng/ml). Survival of mice was monitored over a time period

Techniques Used: Mutagenesis, Mouse Assay, Transfection, Enzyme-linked Immunosorbent Assay

Processing of protein prM after in vitro protease treatment. BHK-21 cells were transfected with either wild-type and prM(ΔR88) (A), prM(ΔR88/S85F) and prM(ΔR88/R89H) (B), or prM(ΔR88/S85F/R86S/R89S) and prM(ΔR88/R86S/R89H)
Figure Legend Snippet: Processing of protein prM after in vitro protease treatment. BHK-21 cells were transfected with either wild-type and prM(ΔR88) (A), prM(ΔR88/S85F) and prM(ΔR88/R89H) (B), or prM(ΔR88/S85F/R86S/R89S) and prM(ΔR88/R86S/R89H)

Techniques Used: In Vitro, Transfection

Activation of mutant viruses by exogenous proteases. BHK-21 cells were transfected with the indicated constructs, and either trypsin or chymotrypsin was added to the medium to a final concentration of 5 μg ml −1 . After 72 h, supernatants
Figure Legend Snippet: Activation of mutant viruses by exogenous proteases. BHK-21 cells were transfected with the indicated constructs, and either trypsin or chymotrypsin was added to the medium to a final concentration of 5 μg ml −1 . After 72 h, supernatants

Techniques Used: Activation Assay, Mutagenesis, Transfection, Construct, Concentration Assay

RNA export kinetics of cleavage site mutants. The amount of viral RNA in cell culture supernatants of BHK-21 cells transfected with the indicated constructs was determined by real-time PCR. Values obtained for mock-transfected cells were below the cutoff
Figure Legend Snippet: RNA export kinetics of cleavage site mutants. The amount of viral RNA in cell culture supernatants of BHK-21 cells transfected with the indicated constructs was determined by real-time PCR. Values obtained for mock-transfected cells were below the cutoff

Techniques Used: Cell Culture, Transfection, Construct, Real-time Polymerase Chain Reaction

26) Product Images from "A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization"

Article Title: A Single-Amino-Acid Polymorphism in Chikungunya Virus E2 Glycoprotein Influences Glycosaminoglycan Utilization

Journal: Journal of Virology

doi: 10.1128/JVI.03116-13

Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P
Figure Legend Snippet: Kinetics of inhibition of 181/25 by heparan sulfate and ammonium chloride. (A) 181/25 virions (MOI of ∼2.5 PFU/cell) were adsorbed to BHK-21 cells at 37°C. At the times shown prior to or during adsorption, heparan sulfate (250 μg/ml) was added to the virus inoculum. After 2 h adsorption, unbound virus was removed, and cells were incubated with medium containing 20 mM NH 4 Cl. At 18 hpi, infected cells were detected by indirect immunofluorescence. Results are expressed as the mean percentage of infected cells normalized to BSA-treated controls from two independent experiments performed in triplicate. Error bars indicate SEM. *, P

Techniques Used: Inhibition, Adsorption, Incubation, Infection, Immunofluorescence

27) Product Images from "Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿"

Article Title: Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct ▿

Journal:

doi: 10.1128/JVI.01017-07

Selection of adaptive mutations in BHK-21 cells.
Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

Techniques Used: Selection

Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,
Figure Legend Snippet: Selection of variants in BHK-21 cells. (A) TBEV-bc (gray bars) and TBEV-wt (black bars) were passaged six times in cell culture, and individual endpoints were determined after each passage by limiting dilution. In the representative experiment shown here,

Techniques Used: Selection, Cell Culture

Selection of adaptive mutations in BHK-21 cells.
Figure Legend Snippet: Selection of adaptive mutations in BHK-21 cells.

Techniques Used: Selection

28) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

29) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

30) Product Images from "Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus"

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus

Journal: Viruses

doi: 10.3390/v10050226

Effects of mutations in SH3 domain ligands on CHIKV RNA infectivity and CHIKV replication. ( A ) BHK-21 cells were electroporated with 1 μg of in vitro transcribed RNAs of CHIKV (wt or bearing mutation in nsP3 HVD); the infectivity of the transcripts was analysed by ICA. Mutant virus RNA infectivity is presented as a percentage of wild-type virus RNA, mean values together with standard error of three independent experiments are shown; ( B ) Growth curve of wt CHIKV or its mutant variants in BHK-21 cells infected at MOI of 0.1. Mean values of four replicates with standard deviation are shown. * denotes p
Figure Legend Snippet: Effects of mutations in SH3 domain ligands on CHIKV RNA infectivity and CHIKV replication. ( A ) BHK-21 cells were electroporated with 1 μg of in vitro transcribed RNAs of CHIKV (wt or bearing mutation in nsP3 HVD); the infectivity of the transcripts was analysed by ICA. Mutant virus RNA infectivity is presented as a percentage of wild-type virus RNA, mean values together with standard error of three independent experiments are shown; ( B ) Growth curve of wt CHIKV or its mutant variants in BHK-21 cells infected at MOI of 0.1. Mean values of four replicates with standard deviation are shown. * denotes p

Techniques Used: Infection, In Vitro, Mutagenesis, Standard Deviation

31) Product Images from "Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses"

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0004163

Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.
Figure Legend Snippet: Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

Techniques Used: Mouse Assay, Infection, Construct, Expressing, Luciferase, Adsorption

32) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

33) Product Images from "A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination ▿"

Article Title: A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination ▿

Journal: Journal of Virology

doi: 10.1128/JVI.01063-09

Detection of recombinants using the recombination trap. (A) Northern blot analysis of intracellular RNA. For TBEV, total cellular RNA was isolated from BHK-21 cells infected with wild-type TBEV (lane 1), mock-transfected cells (lane 2), cells infected
Figure Legend Snippet: Detection of recombinants using the recombination trap. (A) Northern blot analysis of intracellular RNA. For TBEV, total cellular RNA was isolated from BHK-21 cells infected with wild-type TBEV (lane 1), mock-transfected cells (lane 2), cells infected

Techniques Used: Northern Blot, Isolation, Infection, Transfection

Packaging and propagation of complementing TBEV replicons. On the left are fluorescence micrographs showing eGFP and E protein expression in BHK-21 cells 24 h after transfection with in vitro-transcribed full-length genomic RNA or replicon RNA. From top
Figure Legend Snippet: Packaging and propagation of complementing TBEV replicons. On the left are fluorescence micrographs showing eGFP and E protein expression in BHK-21 cells 24 h after transfection with in vitro-transcribed full-length genomic RNA or replicon RNA. From top

Techniques Used: Fluorescence, Expressing, Transfection, In Vitro

Analysis of foci. (A) Typical foci produced in BHK-21 cells after infection with culture supernatants containing wild-type TBEV (left) or a mixture of packaged TBEV replicons ΔC and ΔME-eGFP (right). (B) Immunofluorescence staining of
Figure Legend Snippet: Analysis of foci. (A) Typical foci produced in BHK-21 cells after infection with culture supernatants containing wild-type TBEV (left) or a mixture of packaged TBEV replicons ΔC and ΔME-eGFP (right). (B) Immunofluorescence staining of

Techniques Used: Produced, Infection, Immunofluorescence, Staining

Spiking experiment to examine competition between replicons and full-length virus genomes. (A) Northern blot analysis of total intracellular RNA extracted from BHK-21 cells transfected with full-length TBEV genomic RNA (lane 1), TBEV replicon ΔME
Figure Legend Snippet: Spiking experiment to examine competition between replicons and full-length virus genomes. (A) Northern blot analysis of total intracellular RNA extracted from BHK-21 cells transfected with full-length TBEV genomic RNA (lane 1), TBEV replicon ΔME

Techniques Used: Northern Blot, Transfection

34) Product Images from "Castanospermine, a Potent Inhibitor of Dengue Virus Infection In Vitro and In Vivo"

Article Title: Castanospermine, a Potent Inhibitor of Dengue Virus Infection In Vitro and In Vivo

Journal:

doi: 10.1128/JVI.79.14.8698-8706.2005

Inhibition of viral spread in flaviviruses after treatment with castanospermine. (A) BHK-21 cells were infected with DEN isolates representing each of the four serotypes at an MOI of 0.01 in the presence or absence of 50 μM castanospermine. The
Figure Legend Snippet: Inhibition of viral spread in flaviviruses after treatment with castanospermine. (A) BHK-21 cells were infected with DEN isolates representing each of the four serotypes at an MOI of 0.01 in the presence or absence of 50 μM castanospermine. The

Techniques Used: Inhibition, Infection

(A and B) Inhibition of DEN-2 production in BHK-21 cells. BHK-21 cells were infected at a range of MOI from 0.01 to 10 with DEN-2 (strain 16681) in the absence or presence of increasing concentrations of castanospermine. The amount of virus released into
Figure Legend Snippet: (A and B) Inhibition of DEN-2 production in BHK-21 cells. BHK-21 cells were infected at a range of MOI from 0.01 to 10 with DEN-2 (strain 16681) in the absence or presence of increasing concentrations of castanospermine. The amount of virus released into

Techniques Used: Inhibition, Infection

Effect of castanospermine on WNV and DEN secretion and infectivity. BHK-21 cells were infected with WNV or DEN (MOI of 0.1) and treated with castanospermine (0, 100, or 500 μM), and supernatants were harvested and clarified at 48 h after infection.
Figure Legend Snippet: Effect of castanospermine on WNV and DEN secretion and infectivity. BHK-21 cells were infected with WNV or DEN (MOI of 0.1) and treated with castanospermine (0, 100, or 500 μM), and supernatants were harvested and clarified at 48 h after infection.

Techniques Used: Infection

35) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

36) Product Images from "T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody ▿"

Article Title: T Cells Facilitate Recovery from Venezuelan Equine Encephalitis Virus-Induced Encephalomyelitis in the Absence of Antibody ▿

Journal: Journal of Virology

doi: 10.1128/JVI.02545-09

T cells are required for control of infection and recovery in μMT mice. Female μMT mice (7 to 10 weeks old) were treated with depleting antibodies against CD3, CD4, or CD8 or with an isotype control antibody and then inoculated with 10 3 PFU of V3533 by injection in the left rear footpad. Depletion treatments were continued for 25 days postinfection, at which point the experiment was terminated. (A) Representative dot plots of CD3 + splenocytes from each group, taken at day 25 postinfection. (B) Effect of T cell depletions on weight loss following V3533 infection. Data represent means ± standard errors of the results obtained with 4 to 5 animals per group. (C) Infectious virus from tissues harvested 25 days postinfection, assessed by plaque assays on BHK-21 cells. Each data point represents a single animal, with bars indicating the geometric means. *, P
Figure Legend Snippet: T cells are required for control of infection and recovery in μMT mice. Female μMT mice (7 to 10 weeks old) were treated with depleting antibodies against CD3, CD4, or CD8 or with an isotype control antibody and then inoculated with 10 3 PFU of V3533 by injection in the left rear footpad. Depletion treatments were continued for 25 days postinfection, at which point the experiment was terminated. (A) Representative dot plots of CD3 + splenocytes from each group, taken at day 25 postinfection. (B) Effect of T cell depletions on weight loss following V3533 infection. Data represent means ± standard errors of the results obtained with 4 to 5 animals per group. (C) Infectious virus from tissues harvested 25 days postinfection, assessed by plaque assays on BHK-21 cells. Each data point represents a single animal, with bars indicating the geometric means. *, P

Techniques Used: Infection, Mouse Assay, Injection

V3533 induces mild, transient disease followed by clearance in C57BL/6 mice. (A) Female C57BL/6 mice (7 to 10 weeks old) were inoculated with 10 6 PFU of V3533 by injection in the left rear footpad. At the indicated days postinfection (Day P.I.), serum, spleen, brain, and spinal cord samples were collected from V3533-infected mice and homogenized. The amount of infectious virus present in serum, spleen, brain, and spinal cord samples was then quantified by plaque assays of BHK-21 cells. Data are presented as the means ± standard deviations of results pooled from two independent experiments with 3 to 10 animals per time point. Dotted lines represent the limit of detection. (B) Female C57BL/6 mice (7 to 10 weeks old) were inoculated with 10 3 PFU of wild-type VEEV (V3000) or 10 6 PFU of V3533 by injection in the left rear footpad. Mock-infected mice were infected with diluent alone. Mice were weighed daily, with those losing more than 20% of their starting weight being euthanized as required by UNC IACUC regulations. *, P
Figure Legend Snippet: V3533 induces mild, transient disease followed by clearance in C57BL/6 mice. (A) Female C57BL/6 mice (7 to 10 weeks old) were inoculated with 10 6 PFU of V3533 by injection in the left rear footpad. At the indicated days postinfection (Day P.I.), serum, spleen, brain, and spinal cord samples were collected from V3533-infected mice and homogenized. The amount of infectious virus present in serum, spleen, brain, and spinal cord samples was then quantified by plaque assays of BHK-21 cells. Data are presented as the means ± standard deviations of results pooled from two independent experiments with 3 to 10 animals per time point. Dotted lines represent the limit of detection. (B) Female C57BL/6 mice (7 to 10 weeks old) were inoculated with 10 3 PFU of wild-type VEEV (V3000) or 10 6 PFU of V3533 by injection in the left rear footpad. Mock-infected mice were infected with diluent alone. Mice were weighed daily, with those losing more than 20% of their starting weight being euthanized as required by UNC IACUC regulations. *, P

Techniques Used: Mouse Assay, Injection, Infection

Recovery in μMT mice is associated with control of viral replication in the brain and clearance in the spinal cord. Female μMT or Rag1 −/− mice (7 to 10 weeks old) were inoculated with 10 3 PFU of V3533 by injection in the left rear footpad. (A) At the time points indicated, serum, spleen, brain, and spinal cord samples were collected from infected μMT mice and homogenized. The amount of infectious virus present in the serum, spleen, brain, and spinal cord samples was then quantified by plaque assays of BHK-21 cells. Data points represent individual tissue titers pooled from two independent experiments. (B) Tissue titers from infected μMT mice at day 105 postinfection. (C) Comparison of tissue titers between μMT and Rag1 −/− mice. Data are presented as the means ± SEM of titer values from 3 to 4 animals per group. In all cases, dotted lines represent the limit of detection.
Figure Legend Snippet: Recovery in μMT mice is associated with control of viral replication in the brain and clearance in the spinal cord. Female μMT or Rag1 −/− mice (7 to 10 weeks old) were inoculated with 10 3 PFU of V3533 by injection in the left rear footpad. (A) At the time points indicated, serum, spleen, brain, and spinal cord samples were collected from infected μMT mice and homogenized. The amount of infectious virus present in the serum, spleen, brain, and spinal cord samples was then quantified by plaque assays of BHK-21 cells. Data points represent individual tissue titers pooled from two independent experiments. (B) Tissue titers from infected μMT mice at day 105 postinfection. (C) Comparison of tissue titers between μMT and Rag1 −/− mice. Data are presented as the means ± SEM of titer values from 3 to 4 animals per group. In all cases, dotted lines represent the limit of detection.

Techniques Used: Mouse Assay, Injection, Infection

37) Product Images from "Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence"

Article Title: Replicase Complex Genes of Semliki Forest Virus Confer Lethal Neurovirulence

Journal: Journal of Virology

doi:

Viral RNA synthesis during early infection measured by scintillation counting. BHK-21 monolayers (solid bars) and 72-h-cultured rat cerebellar granule neurons (open bars) were infected with parental strains and recombinants at 20 PFU per cell. [ 3 H]uridine was added at 3 h postinfection, and cells were lysed 3 h later in 10% SDS. Actinomycin D was used to inhibit cellular RNA synthesis. Control cells (C) were uninfected. The star indicates a value below 500 cpm.
Figure Legend Snippet: Viral RNA synthesis during early infection measured by scintillation counting. BHK-21 monolayers (solid bars) and 72-h-cultured rat cerebellar granule neurons (open bars) were infected with parental strains and recombinants at 20 PFU per cell. [ 3 H]uridine was added at 3 h postinfection, and cells were lysed 3 h later in 10% SDS. Actinomycin D was used to inhibit cellular RNA synthesis. Control cells (C) were uninfected. The star indicates a value below 500 cpm.

Techniques Used: Infection, Cell Culture

38) Product Images from "Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome"

Article Title: Cloning and Mutagenesis of the Murine Gammaherpesvirus 68 Genome as an Infectious Bacterial Artificial Chromosome

Journal: Journal of Virology

doi:

Comparison of the in vitro growth properties of several recombinant MHV-68 mutants and WT MHV-68. BHK-21 cells were infected at an MOI of 0.1. Cells and supernatants were harvested at the indicated time points, and viral titers were determined by plaque assay. Titers at 0 h represent input inocula. (A) Growth properties of RγHV68A98.01 compared to WT MHV-68; (B) Expression of gfp in NIH3T3 cells infected with RγHV68A98.01; (C) Growth properties of RγHV68A98.02 compared to RγHV68A98.01; (D) Lack of gfp expression in NIH3T3 cells infected with RγHV68A98.02; (E) Growth properties of RγHV68A98.03 and RγHV68A98.04 compared to RγHV68A98.01; (F) Southern blot analysis of Eco RI-digested DNA isolated from cells infected with WT MHV-68 (lane 1), RγHV68A98.01 (lane 2), RγHV68A98.03 (lane 3), and RγHV68A98.04 (lane 4) with a probe specific for the Eco RI K fragment after digestion of the DNA with Eco RI.
Figure Legend Snippet: Comparison of the in vitro growth properties of several recombinant MHV-68 mutants and WT MHV-68. BHK-21 cells were infected at an MOI of 0.1. Cells and supernatants were harvested at the indicated time points, and viral titers were determined by plaque assay. Titers at 0 h represent input inocula. (A) Growth properties of RγHV68A98.01 compared to WT MHV-68; (B) Expression of gfp in NIH3T3 cells infected with RγHV68A98.01; (C) Growth properties of RγHV68A98.02 compared to RγHV68A98.01; (D) Lack of gfp expression in NIH3T3 cells infected with RγHV68A98.02; (E) Growth properties of RγHV68A98.03 and RγHV68A98.04 compared to RγHV68A98.01; (F) Southern blot analysis of Eco RI-digested DNA isolated from cells infected with WT MHV-68 (lane 1), RγHV68A98.01 (lane 2), RγHV68A98.03 (lane 3), and RγHV68A98.04 (lane 4) with a probe specific for the Eco RI K fragment after digestion of the DNA with Eco RI.

Techniques Used: In Vitro, Recombinant, Infection, Plaque Assay, Expressing, Southern Blot, Isolation

39) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

40) Product Images from "Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus"

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

Journal: Journal of Virology

doi: 10.1128/JVI.79.18.11813-11823.2005

Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
Figure Legend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

Techniques Used: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein
Figure Legend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

Techniques Used: Transfection, Mutagenesis

Related Articles

Electroporation:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Full-length RNAs were transcribed in vitro from the plasmids pTNd/prM(ΔR88), pTNd/prM(ΔR88/C79Y), and pTNd/prM(ΔR88/Y76C) using T7 RNA polymerase (Ambion) and then transfected into BHK-21 cells by electroporation (Bio-Rad Gene Pulser apparatus; two pulses at 1.8 kV, 25 μF, and 200 Ω) as described previously ( ). .. BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus
Article Snippet: .. Virus recovery was performed as follows, 8 × 106 BHK-21 cells resuspended in ice-cold PBS were transfected with 10 μg of in vitro transcribed RNAs by electroporation using a Gene Pulser II unit (two pulses at 850 V and 25 μF, Bio-Rad, Hercules, CA, USA), 48 h later virus stock was harvested to be used in further experiments. .. Infectious centre assay (ICA) was conducted to measure RNA infectivity, 1 μg of in vitro transcribed RNA was used and ten-fold dilutions of electroporated cells were seeded on top of 1.5 × 106 BHK-21 cells grown in the wells of a 6-well tissue culture plate.

Transfection:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Full-length RNAs were transcribed in vitro from the plasmids pTNd/prM(ΔR88), pTNd/prM(ΔR88/C79Y), and pTNd/prM(ΔR88/Y76C) using T7 RNA polymerase (Ambion) and then transfected into BHK-21 cells by electroporation (Bio-Rad Gene Pulser apparatus; two pulses at 1.8 kV, 25 μF, and 200 Ω) as described previously ( ). .. BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus
Article Snippet: .. Virus recovery was performed as follows, 8 × 106 BHK-21 cells resuspended in ice-cold PBS were transfected with 10 μg of in vitro transcribed RNAs by electroporation using a Gene Pulser II unit (two pulses at 850 V and 25 μF, Bio-Rad, Hercules, CA, USA), 48 h later virus stock was harvested to be used in further experiments. .. Infectious centre assay (ICA) was conducted to measure RNA infectivity, 1 μg of in vitro transcribed RNA was used and ten-fold dilutions of electroporated cells were seeded on top of 1.5 × 106 BHK-21 cells grown in the wells of a 6-well tissue culture plate.

In Vitro:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Full-length RNAs were transcribed in vitro from the plasmids pTNd/prM(ΔR88), pTNd/prM(ΔR88/C79Y), and pTNd/prM(ΔR88/Y76C) using T7 RNA polymerase (Ambion) and then transfected into BHK-21 cells by electroporation (Bio-Rad Gene Pulser apparatus; two pulses at 1.8 kV, 25 μF, and 200 Ω) as described previously ( ). .. BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

Article Title: Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus
Article Snippet: .. Virus recovery was performed as follows, 8 × 106 BHK-21 cells resuspended in ice-cold PBS were transfected with 10 μg of in vitro transcribed RNAs by electroporation using a Gene Pulser II unit (two pulses at 850 V and 25 μF, Bio-Rad, Hercules, CA, USA), 48 h later virus stock was harvested to be used in further experiments. .. Infectious centre assay (ICA) was conducted to measure RNA infectivity, 1 μg of in vitro transcribed RNA was used and ten-fold dilutions of electroporated cells were seeded on top of 1.5 × 106 BHK-21 cells grown in the wells of a 6-well tissue culture plate.

Isolation:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Viruses isolated from the brains of mice infected with mutants prM(ΔR88/Y76C) and prM(ΔR88/C79Y) were subsequently used to infect BHK-21 cells. .. As observed with the original isolates prM(ΔR88)-A and prM(ΔR88)-B (see above), the recombinant viruses prM(ΔR88/Y76C) and prM(ΔR88/C79Y) recovered from mice exhibited only single-round infectivity when used to infect BHK-21 cells.

Infection:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Viruses isolated from the brains of mice infected with mutants prM(ΔR88/Y76C) and prM(ΔR88/C79Y) were subsequently used to infect BHK-21 cells. .. As observed with the original isolates prM(ΔR88)-A and prM(ΔR88)-B (see above), the recombinant viruses prM(ΔR88/Y76C) and prM(ΔR88/C79Y) recovered from mice exhibited only single-round infectivity when used to infect BHK-21 cells.

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. End-point dilution experiments performed with mouse brain suspensions on BHK-21 cells yielded titers of 1.5 × 102 IU/ml and 1.5 × 104 IU/ml for prM(ΔR88/Y76C) and prM(ΔR88/C79Y), respectively, but supernatants harvested from infected cells were not able to initiate subsequent rounds of infection in BHK-21 cells (not shown). .. In summary, the results indicated that the Cys mutations in the “pr” part of protein prM did indeed confer viability to the furin-deficient cleavage mutant in suckling mice, but not in BHK-21 cells.

Purification:

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses
Article Snippet: .. For virus recovery, two μg of purified plasmid was electroporated into BHK-21 cells using a BioRad Gene Pulser (Hercules, CA). .. Briefly, 80–90% confluent T175 flasks of BHK-21 cells were trypsinized and washed twice in PBS, followed by one wash in cytomix buffer [ ].

other:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: This impairment, however, is not sufficient to explain the inability to propagate these viruses in BHK-21 cells at 37°C because passages were successful at 28°C (Fig. ), even though export was impaired at least as much at 28°C as it was at 37°C.

Mouse Assay:

Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus
Article Snippet: .. Viruses isolated from the brains of mice infected with mutants prM(ΔR88/Y76C) and prM(ΔR88/C79Y) were subsequently used to infect BHK-21 cells. .. As observed with the original isolates prM(ΔR88)-A and prM(ΔR88)-B (see above), the recombinant viruses prM(ΔR88/Y76C) and prM(ΔR88/C79Y) recovered from mice exhibited only single-round infectivity when used to infect BHK-21 cells.

Plasmid Preparation:

Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses
Article Snippet: .. For virus recovery, two μg of purified plasmid was electroporated into BHK-21 cells using a BioRad Gene Pulser (Hercules, CA). .. Briefly, 80–90% confluent T175 flasks of BHK-21 cells were trypsinized and washed twice in PBS, followed by one wash in cytomix buffer [ ].

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    Bio-Rad bhk 21 cells
    Immunofluorescence staining of protein E in <t>BHK-21</t> cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
    Bhk 21 Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bhk 21 cells - by Bioz Stars, 2020-09
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    Bio-Rad bhk 21 transfected cells
    ( A). Schematic representation of the FMDV viral genome . A gray line at the 5´end depicts structural elements, including the S hairpin, the polyC tract (C n ), 2–4 pseudoknots (Pk), and the cis replicative element ( cre) , followed by the IRES element depicted by a solid black line. A solid gray rectangle represents the viral polyprotein with indication of the region encoding the capsid protein VP1. The 3´UTR consists of 2 stem-loops followed by a poly(A) tail (A n ). ( B ) Analysis of virus multiplication in the presence of IRAB. Viral yield reduction induced by increasing amounts of IRAB (black bars) or Isis-11 (striped bars). In vitro synthesized FMDV RNA (50 pg) was <t>transfected</t> in IRAB treated <t>BHK-21</t> cell monolayers, in duplicate wells. Virus yield was determined using fresh cells monolayers as the number of plaque forming units (PFU)/ml in the supernatant 24 hpt, shown relative to the control RNA which was set at 100%. Values represent the mean and standard deviation of triplicate assays. P values were calculated using two-tail Student´s t -test; differences were considered significant when P
    Bhk 21 Transfected Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 transfected cells/product/Bio-Rad
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    bhk 21 transfected cells - by Bioz Stars, 2020-09
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    Image Search Results


    Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

    Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Transfection, Mutagenesis

    Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Identifying the Role of E2 Domains on Alphavirus Neutralization and Protective Immune Responses

    doi: 10.1371/journal.pntd.0004163

    Figure Lengend Snippet: Protection and neutralizing antibody response elicited by chimeric CHIKV/SFV. Adult C57bl/6 mice (n = 6) were infected with 10 5 PFU of CHIK, SFV, or chimeric viruses (ΔDomA, ΔDomB or ΔDomC) in the left hind footpad. Two months later, mice were bled for neutralizing antibodies and challenged with 10 5 PFU SFV. A) Levels of neutralizing antibodies against SFV were measured by incubating serum from vaccinated mice with a SFV construct expressing nano-luciferase overnight at 4°C. The next day, the virus:serum mixture was used to infect BHK-21 cells in 96 well plates. After one hour adsorption period, cells were washed and fresh media was added. After five hours infection, cells were lysed and luciferase signal measured. Relative luminescence was normalized to a mock vaccinated control serum. SFV is not included as all infected mice rapidly succumbed to infection. B) Challenged mice were monitored for 15 days following infection. Data are expressed as percent survival.

    Article Snippet: For virus recovery, two μg of purified plasmid was electroporated into BHK-21 cells using a BioRad Gene Pulser (Hercules, CA).

    Techniques: Mouse Assay, Infection, Construct, Expressing, Luciferase, Adsorption

    ( A). Schematic representation of the FMDV viral genome . A gray line at the 5´end depicts structural elements, including the S hairpin, the polyC tract (C n ), 2–4 pseudoknots (Pk), and the cis replicative element ( cre) , followed by the IRES element depicted by a solid black line. A solid gray rectangle represents the viral polyprotein with indication of the region encoding the capsid protein VP1. The 3´UTR consists of 2 stem-loops followed by a poly(A) tail (A n ). ( B ) Analysis of virus multiplication in the presence of IRAB. Viral yield reduction induced by increasing amounts of IRAB (black bars) or Isis-11 (striped bars). In vitro synthesized FMDV RNA (50 pg) was transfected in IRAB treated BHK-21 cell monolayers, in duplicate wells. Virus yield was determined using fresh cells monolayers as the number of plaque forming units (PFU)/ml in the supernatant 24 hpt, shown relative to the control RNA which was set at 100%. Values represent the mean and standard deviation of triplicate assays. P values were calculated using two-tail Student´s t -test; differences were considered significant when P

    Journal: RNA Biology

    Article Title: Local RNA flexibility perturbation of the IRES element induced by a novel ligand inhibits viral RNA translation

    doi: 10.1080/15476286.2015.1025190

    Figure Lengend Snippet: ( A). Schematic representation of the FMDV viral genome . A gray line at the 5´end depicts structural elements, including the S hairpin, the polyC tract (C n ), 2–4 pseudoknots (Pk), and the cis replicative element ( cre) , followed by the IRES element depicted by a solid black line. A solid gray rectangle represents the viral polyprotein with indication of the region encoding the capsid protein VP1. The 3´UTR consists of 2 stem-loops followed by a poly(A) tail (A n ). ( B ) Analysis of virus multiplication in the presence of IRAB. Viral yield reduction induced by increasing amounts of IRAB (black bars) or Isis-11 (striped bars). In vitro synthesized FMDV RNA (50 pg) was transfected in IRAB treated BHK-21 cell monolayers, in duplicate wells. Virus yield was determined using fresh cells monolayers as the number of plaque forming units (PFU)/ml in the supernatant 24 hpt, shown relative to the control RNA which was set at 100%. Values represent the mean and standard deviation of triplicate assays. P values were calculated using two-tail Student´s t -test; differences were considered significant when P

    Article Snippet: Western blot analysis Equal amounts of total protein prepared from BHK-21 transfected cells were resolved in 10% SDS-PAGE, transferred to a PVDF membrane (Bio-Rad).

    Techniques: In Vitro, Synthesized, Transfection, Standard Deviation