Journal: Pathogens

Article Title: A Virus-Like Particle-Based Vaccine Candidate against the Tick-Borne Powassan Virus Induces Neutralizing Antibodies in a Mouse Model

doi: 10.3390/pathogens10060680

Figure Lengend Snippet: Analysis of POWV EDIII recombinant protein by SDS-PAGE and Western blot. Coomassie blue staining indicated electrophoretic mobility of approximately 12.7 kDa (left panel 1). Western blot analysis of POWV EDIII and ZIKV EDIII control (panels 2 to 4 from left). The anti-histidine monoclonal antibody (Anti-His MAB) detected both recombinant proteins (panel 2). Anti-POWV ascitic fluid antibody (POWV AF) (panel 3) and LGTV anti-envelope protein monoclonal antibody (LGTV E MAB) (panel 4) reacted specifically with POWV EDIII and not with ZIKV EDIII.

Article Snippet: Primary and secondary antibodies used for the immunoassays included Powassan virus immune ascitic fluid [V-518-711-562] (ATCC ® , Manassas, VA, USA) (Cat. VR-1262AFTM), monoclonal anti-Langat virus envelope glycoprotein (E), clone 10F6 (BEI Resources, Manassas, VA, USA) (Cat. NR-40316), Powassan virus prM protein antibody (GeneTex, Irvine, CA, USA) (Cat. GTX132055), His-Tag antibody (H-3) (Santa Cruz Biotechnology, Dallas, TX, USA) (Cat. sc-8036), goat anti-mouse IgG antibody, and HRP conjugate (ImmunoReagents, Raleigh, NC, USA) (Cat. GtxMu-003-DHRPX).

Techniques: Recombinant, SDS Page, Western Blot, Staining, Control

Journal: Pathogens

Article Title: A Virus-Like Particle-Based Vaccine Candidate against the Tick-Borne Powassan Virus Induces Neutralizing Antibodies in a Mouse Model

doi: 10.3390/pathogens10060680

Figure Lengend Snippet: Production of POW-VLP in mammalian cells. ( A ) The expressed recombinant E protein used for POW-VLP generation undergoes processing and self-assembly into virus-like particles. ( B ) Indirect immunofluorescence detection of the POWV E protein expressed in transfected mammalian cells. ( C ) Purified POW-VLPs were detected by Western blot analysis from cell culture supernatants and the cytosolic extract of transfected mammalian cells. POW-VLPs were poorly secreted and were primarily retained in the cytosol. Both assays were performed using anti-POWV ascitic fluid antibodies.

Article Snippet: Primary and secondary antibodies used for the immunoassays included Powassan virus immune ascitic fluid [V-518-711-562] (ATCC ® , Manassas, VA, USA) (Cat. VR-1262AFTM), monoclonal anti-Langat virus envelope glycoprotein (E), clone 10F6 (BEI Resources, Manassas, VA, USA) (Cat. NR-40316), Powassan virus prM protein antibody (GeneTex, Irvine, CA, USA) (Cat. GTX132055), His-Tag antibody (H-3) (Santa Cruz Biotechnology, Dallas, TX, USA) (Cat. sc-8036), goat anti-mouse IgG antibody, and HRP conjugate (ImmunoReagents, Raleigh, NC, USA) (Cat. GtxMu-003-DHRPX).

Techniques: Recombinant, Virus, Immunofluorescence, Transfection, Purification, Western Blot, Cell Culture

Journal: Pathogens

Article Title: A Virus-Like Particle-Based Vaccine Candidate against the Tick-Borne Powassan Virus Induces Neutralizing Antibodies in a Mouse Model

doi: 10.3390/pathogens10060680

Figure Lengend Snippet: Characterization of POW-VLPs. ( A ) POW-VLP antigenicity assessed by Western blot analysis. Anti-POWV ascitic fluid antibodies (POWV AF) (panel 1) and LGTV anti-envelope protein monoclonal antibodies (LGTV E MAB) (panel 2) recognized E protein from POW-VLP; the presence of prM protein from POW-VLPs was detected using POWV anti-prM antibody (panel 3). ( B ) Transmission electron microscopy analysis indicated icosahedral morphology and size of 40–50 nm for POW-VLP.

Article Snippet: Primary and secondary antibodies used for the immunoassays included Powassan virus immune ascitic fluid [V-518-711-562] (ATCC ® , Manassas, VA, USA) (Cat. VR-1262AFTM), monoclonal anti-Langat virus envelope glycoprotein (E), clone 10F6 (BEI Resources, Manassas, VA, USA) (Cat. NR-40316), Powassan virus prM protein antibody (GeneTex, Irvine, CA, USA) (Cat. GTX132055), His-Tag antibody (H-3) (Santa Cruz Biotechnology, Dallas, TX, USA) (Cat. sc-8036), goat anti-mouse IgG antibody, and HRP conjugate (ImmunoReagents, Raleigh, NC, USA) (Cat. GtxMu-003-DHRPX).

Techniques: Western Blot, Bioprocessing, Transmission Assay, Electron Microscopy

Journal: Pathogens

Article Title: A Virus-Like Particle-Based Vaccine Candidate against the Tick-Borne Powassan Virus Induces Neutralizing Antibodies in a Mouse Model

doi: 10.3390/pathogens10060680

Figure Lengend Snippet: Neutralization test for POW-VLP immunization in a murine model. The reporter virus-particle neutralization test demonstrated that POW-VLPs induce a dose-dependent neutralization activity in a murine model (* p < 0.05; ** p < 0.005; *** p < 0.0005). Neutralization activity quantification is indicated by 50% inhibition of plaque reduction neutralization test (PRNT 50 ). Anti-POWV ascitic fluid antibody was included as a control (POWV AF).

Article Snippet: Primary and secondary antibodies used for the immunoassays included Powassan virus immune ascitic fluid [V-518-711-562] (ATCC ® , Manassas, VA, USA) (Cat. VR-1262AFTM), monoclonal anti-Langat virus envelope glycoprotein (E), clone 10F6 (BEI Resources, Manassas, VA, USA) (Cat. NR-40316), Powassan virus prM protein antibody (GeneTex, Irvine, CA, USA) (Cat. GTX132055), His-Tag antibody (H-3) (Santa Cruz Biotechnology, Dallas, TX, USA) (Cat. sc-8036), goat anti-mouse IgG antibody, and HRP conjugate (ImmunoReagents, Raleigh, NC, USA) (Cat. GtxMu-003-DHRPX).

Techniques: Neutralization, Virus, Activity Assay, Inhibition, Plaque Reduction Neutralization Test, Control

Journal: Vaccines

Article Title: Dynamics of Whole Virus and Non-Structural Protein 1 (NS1) IgG Response in Mice Immunized with Two Commercial Tick-Borne Encephalitis Vaccines

doi: 10.3390/vaccines10071001

Figure Lengend Snippet: ( A ) Experimental protocol. Mice were immunized with either Encepur or FSME-Immun vaccines. Control mice received only the adjuvant. A total of six doses of vaccine were administered two weeks apart. Blood samples were collected from the tail vein of the mice 7 days after each vaccination. (Figure created using Servier Medical Art, available at www.servier.com , accessed on 1 April 2021). ( B ) Dynamics of whole virus (WV)-specific IgG in mice vaccinated with either Encepur or FSME-Immun. Mice receiving only the adjuvant were used as controls. Concentrations of anti-WV IgG in vaccinated mice were statistically compared with unvaccinated controls. ( C ) Comparison of WV-specific IgG levels in mice vaccinated with six doses of Encepur or FSME-Immun vaccine. ( D ) Dynamics of NS1-specific IgG in mice immunized with either Encepur or FSME-Immun vaccines. Mice receiving adjuvant only were used as controls. Concentrations of anti-NS1 IgG in vaccinated mice were statistically compared with unvaccinated controls. **, p < 0.01.

Article Snippet: Detection of anti-NS1 specific IgG antibodies in the sera tested was performed using the Mouse Anti-Tick-Borne Encephalitis virus NS1 IgG Elisa Kit (Alpha Diagnostic International, San Antonio, TX, USA) according to the manufacturer’s instructions.

Techniques: Vaccines, Control, Adjuvant, Virus, Comparison

Journal: PLoS Pathogens

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

doi: 10.1371/journal.ppat.1001255

Figure Lengend Snippet: SW13 (A, B) or Huh7.5 (C–E) cells were seeded in equal numbers into plates and transduced with V1 vector expressing GFP (GFP, filled circles), V1 vector expressing full-length human DNAJC14 (FL, open circles) or V1 vector expressing truncated (aa 305–702) human DNAJC14 (NT1, triangles). After 2 days, the cells were challenged (moi = 5) with YFV 17D (A), YFV Asibi (B), Kunjin virus (C), Langat virus (D) or were challenged (moi = 0.1) with HCV Jc1FLAG2/p7-nsGluc2A. The medium (A–D) or cells (E) were harvested at the indicated times for quantification of virus replication. For A and B, a single separate well was utilized for each time point, and virion production was enumerated by plaque assay. In both cases, there were fewer than 100 plaque-forming units at 12 h post infection. The dashed line indicates the sensitivity of the plaque assay. Pfu, plaque forming units. For C and D, duplicate wells were infected and the medium was harvested and replaced at each timepoint. Virion production since the prior time point was enumerated by focus forming assay as described in . Data points represent the mean titer; error bars indicate the range. Similar results were obtained in an independent experiment for both Kunjin and Langat viruses. FFU, focus forming units. For E, cells were harvested at the indicated times after infection for measurement of luciferase activity as described in . Data points represent mean values obtained from triplicate wells; error bars indicate the standard deviation. RLU, relative light units.

Article Snippet: After 4 d the monolayers were fixed with 100% methanol and plaques were visualized by incubation with polyclonal mouse antibody cross-reactive to Langat (hyperimmune mouse ascites fluid, clone Russian Spring Summer Encephalitis VR79; ATCC) or polyclonal mouse anti-West Nile virus E protein (obtained from Dr. Robert Tesh, World Reference Center for Emerging Viruses and Arboviruses) followed by secondary goat anti-mouse peroxidase-labelled polymer (DAKO Envision Systems) and application of peroxidase substrate containing 0.4 mg/ml 3,3′ diaminobenzidine and 0.0135% hydrogen peroxide in PBS.

Techniques: Transduction, Plasmid Preparation, Expressing, Virus, Plaque Assay, Infection, Focus Forming Assay, Luciferase, Activity Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

doi: 10.1371/journal.ppat.1001255

Figure Lengend Snippet: (A) SW13 cells were left untransduced (top row) or were transduced (lower 2 rows) with the noninhibitory V1-hDNAJC14 mutants FL-H471Q or CT1 as indicated. Two d later the cells were mock treated (left panels) or were challenged with YFV (moi = 5, right 3 panels). After an additional 2 d, the cells were fixed and immunostained with rabbit anti-YFV NS3 polyclonal antibodies (NS3), and mouse anti-calnexin antibody (calnexin) or mouse anti-myc monoclonal antibody (myc) as indicated. AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG antibodies were used as secondary antibodies. The cells were analyzed by confocal microscopy and representative images are shown. Calnexin or DNAJC14 mutants are shown in green, YFV NS3 is shown in red, and the merged images are shown on the right. (B) SW13 cells were left untransduced or were transduced with the V1-hDNAJC14-CT1 mutant (CT1-myc) as indicated and were infected 2 d later with YFV (moi = 1). After 2 d of infection, myc-tagged DNAJC14-CT1 was immunoprecipitated using anti-myc antibody. Western blots were performed using antibodies against NS3, calnexin and the myc epitope tag as indicated. (C) SW13 cells were left uninfected or were infected with YFV (moi = 1) as indicated. The cells were fixed 1 d later and analyzed by confocal microscopy for endogenous DNAJC14 (red) and double stranded RNA (dsRNA, green). The merged image is shown on the right. Arrows indicate several areas of colocalized DNAJC14 and dsRNA.

Article Snippet: After 4 d the monolayers were fixed with 100% methanol and plaques were visualized by incubation with polyclonal mouse antibody cross-reactive to Langat (hyperimmune mouse ascites fluid, clone Russian Spring Summer Encephalitis VR79; ATCC) or polyclonal mouse anti-West Nile virus E protein (obtained from Dr. Robert Tesh, World Reference Center for Emerging Viruses and Arboviruses) followed by secondary goat anti-mouse peroxidase-labelled polymer (DAKO Envision Systems) and application of peroxidase substrate containing 0.4 mg/ml 3,3′ diaminobenzidine and 0.0135% hydrogen peroxide in PBS.

Techniques: Confocal Microscopy, Transduction, Mutagenesis, Infection, Immunoprecipitation, Western Blot