Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Schematic representation of the T-DNA region of the pBYR2e RSV-F Fc fusion plant expression vector. The T-DNA region plays a crucial role in facilitating the transfer of the gene of interest into plant cells. It includes the left and right borders, known as RB and LB, which serve as the boundaries for gene transfer. The Pin II 3′ sequence derived from potato proteinase inhibitor II acts as a border element. The vector also incorporates several important components, such as the Tomato Bushy Stunt Virus (TBSV) RNA silencing suppressor, P19; the Cauliflower Mosaic Virus (CaMV) 35s promoter, P35s; the CaMV enhancer, P35s × 2; the tobacco extension gene region, Ext3′ FL, 3′; the tobacco RB7 promoter, Rb7 5′ del; the Bean Yellow Dwarf Virus (BeYDV) short intergenic region, SIR; the BeYDV long intergenic region, LIR; and the BeYDV replication initiation proteins, Rep and RepA, along with C2/C1.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Virus

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: SDS-PAGE and western blot analysis of plant-produced RSV-F Fc fusion protein. SDS-PAGE stained with Instant Blue™ under non-reducing (a) and reducing condition (b) whereas, lane M, Protein ladder; lane 1, purified RSV F-Native Fc fusion protein; lane 2, purified SC-TM Fc fusion protein. Western blot analysis under non-reducing conditions (c) and reducing condition (d), the membrane was probed with anti-human IgG Fc HRP. The arrowhead indicates the major band.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: SDS Page, Western Blot, Produced, Staining, Purification, Membrane

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: The binding activity of the plant-produced RSV-F Fc fusion protein to Motavizumab (anti-RSV mAb) was analyzed by ELISA. The plant-produced RBD-Fc was used as a negative control. Data are presented as mean ± SD of triplicates.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Binding Assay, Activity Assay, Produced, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Schematic illustration of immunization protocol and blood collection. Groups of mice (five mice per group) were intramuscularly immunized with 5 µg of RSV-F Fc fusion protein with 3M-SE or 3M-Alum adjuvant. A booster dose was administered 21 days after the first immunization. Mice sera were collected on day 0 (pre-immune sera) and day 35 post-immunization.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Adjuvant

Journal: Biotechnology Reports

Article Title: Immunogenicity of a recombinant plant-produced respiratory syncytial virus F subunit vaccine in mice

doi: 10.1016/j.btre.2023.e00826

Figure Lengend Snippet: Immunogenicity of RSV-F Fc fusion protein in mice. The mice sera was collected on day 35 and RSV specific titer was analyzed by ELISA using RSV-F his protein as the capture antigen. Data were presented as GMT ± 95 % CI of the endpoint titer ( n = 5). A nonparametric test was used to compare each group. (∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001).

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti RSV-F mAb (Sino Biological, Cat#11049-R302, Beijing, China) diluted 1:3000 in 1x PBS containing 0.5 % BSA and 0.01 % Tween-20 added to each well and incubated for 1 h at 37 °C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IM immunization with MF59/preF vaccine induced systemic humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intramuscularly with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and ILN were harvested on day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intramuscularly immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–G) The representative images and quantitative analysis of preF‐specific IgG + ASCs in bone marrow (E), spleen (F), and lung (G). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–G. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–G. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IN immunization with MF59/preF vaccine induced local humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intranasally with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intranasally immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–J) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (E, H), spleen (F, I), and lung (G, J). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–J. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–J. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: Combination of IM and IN immunization using MF59/preF vaccine elicited both local and systemic humoral and B‐cell immune responses. (A–B) The schematic representation of the mouse immunization and sample collection protocol. BALB/c mice received two intramuscular doses of the MF59/preF vaccine followed by a single intranasal dose (IM‐IM‐IN), or one intramuscular dose followed by two intranasal doses (IM‐IN‐IN). Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (C) Endpoint titers of anti‐preF/postF IgG in sera on Day 56. (D) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (E) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (F‐K) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (F, I), spleen (G, J), and lung (H, K). Data are presented as geometric mean values ± SD in C–E. The middle line indicates the median while the whisker shows the data range in F–K. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in C–K. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: Human Vaccines & Immunotherapeutics

Article Title: Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana

doi: 10.1080/21645515.2024.2327142

Figure Lengend Snippet: Schematic representation of the T-DNA region of the pBYR2e RSV-F Fc fusion plant expression vector. The T-DNA region plays a crucial role in facilitating the transfer of the gene of interest into plant cells. It includes the left border (LB) and right border (RB) which serve as the boundaries for gene transfer. The Pin II 3’ sequence derived from potato proteinase inhibitor II acts as a border element helping to facilitate the insertion of the desired genes into the plant genome. The vector also incorporates several important components, such as the Tomato Bushy Stunt Virus (TBSV) RNA silencing suppressor, P19; the Cauliflower Mosaic Virus (CaMV) 35s promoter, P35s; the CaMV enhancer, P35s×2; the tobacco extension gene region, Ext3’ FL, 3‘; the tobacco RB7 promoter, Rb7 5’ del; the Bean Yellow Dwarf Virus (BeYDV) short intergenic region, SIR; the BeYDV long intergenic region, LIR; and the BeYDV replication initiation proteins, Rep and RepA, along with C2/C1. .

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti-RSV-F mAb (Cat:11049-R302-H, Sino Biological, Beijing, China) diluted 1:3000 in PBS containing 0.5% BSA and 0.01% Tween-20 added to each well and incubated for 1 h at 37°C.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Virus

Journal: Human Vaccines & Immunotherapeutics

Article Title: Neutralizing activity of anti-respiratory syncytial virus monoclonal antibody produced in Nicotiana benthamiana

doi: 10.1080/21645515.2024.2327142

Figure Lengend Snippet: Binding characteristics of plant-produced anti-RSV mAbs. ELISA was used to quantify the specific binding to the RSV-F his-tagged monomer protein. The purified plant produced anti-RSV mAbs, and the plant produced nivolumab (anti PD-1, as a negative control). The binding activity of the antibody was detected with HRP-conjugated anti-human IgG antibody. The data are shown as the mean of triplicates and error bars represent standard deviation. The EC 50 was calculated by using GraphPad Prism software 9.3.

Article Snippet: Human RSV fusion glycoprotein was detected with rabbit anti-RSV-F mAb (Cat:11049-R302-H, Sino Biological, Beijing, China) diluted 1:3000 in PBS containing 0.5% BSA and 0.01% Tween-20 added to each well and incubated for 1 h at 37°C.

Techniques: Binding Assay, Produced, Enzyme-linked Immunosorbent Assay, Purification, Negative Control, Activity Assay, Standard Deviation, Software

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Respiratory syncytial virus spreading in culture in inhibited by Ginkgolic acid. ( A ) Cell cytotoxicity of Ginkgolic acid in Vero E6 cells. Cells were treated with Ginkgolic acid in a two-fold dilution series starting 50µM, for 5 days. CC 50 value was assessed based on cell viability percentage compared to DMSO treated cells. ( B ) Flow cytometry 24 h post Ginkgolic acid treatment of Vero E6 cells infected with hRSV-mKate2. Cells were infected for 2 h with hRSV-mKate2 at a MOI of 0.05. Post infection the cells were washed 3 times with PBS and media containing DMSO of Ginkgolic acid at the indicated concentrations were added to the cultures for 24 h. The percentage of cells scoring red-positive relative to DMSO control (set as 100) are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( C ) hRSV-mKate2 spread in Vero E6 cultures post Ginkgolic acid treatment. Cells were infected as in (B) and were daily monitored for the spread of hRSV-mKate2 using fluorescence microscopy to image the presence of the mKate2 fluorescent reporter. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm.

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Virus, Flow Cytometry, Infection, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Infectivity of respiratory syncytial virus is inhibited post Ginkgolic acid treatment. ( A ) Cell cytotoxicity of Ginkgolic acid in A549 cells. Cells were treated with Ginkgolic acid in a two-fold dilution series starting 50 µM, for 96 h. CC 50 value was assessed based on cell viability percentage compared to DMSO treated cells. The data are mean ± SEM from three independent experiments (n = 3). Fluorescence microscopy monitoring the expression of mKate2 fluorescent protein in A549 cells ( B ) or Vero E6 ( C ) infected for 24 h with hRSV-mKate2 viruses that were pre-treated for 1 h with DMSO or the indicated Ginkgolic acid concentration. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm. ( D ) Flow cytometry of cells from ( B ) and ( C ) was used to determine the number of mKate2 expressing cells in each cultures and calculate the IC 50 values of Ginkgolic acid. The data are mean ± SEM from three independent experiments (n = 3).

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Infection, Virus, Fluorescence, Microscopy, Expressing, Concentration Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Ginkgolic acid inhibits orthopneumo- and metapneumo- virus infectivity

doi: 10.1038/s41598-024-58032-8

Figure Lengend Snippet: Respiratory syncytial virus assembly and release are not affected by Ginkgolic acid treatment. A549 cells were infected with hRSV-mKate2 for 2 h, after which the cells were washed with PBS and media containing DMSO or GA at various concentration were added to the cultures for 24 h. ( A ) Fluorescence microscopy to detect the expression of mKate2 fluorescent protein in infected and treated cells. Shown are representative fields of view from a single experiment out of three independent experiments (n = 3). B.F, brightfield. Scale bar, 100 μm. ( B ) Flow cytometry of cells from (A) to quantify the number of mKate2 expressing cells. The percent cells scoring red-positive relative to DMSO control (set as 100) are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( C ) hRSV Nucleocapsid expression levels from infected and treated cell lysates were assessed using western blot with specific anti-hRSV Nucleocapsid antibody. Shown are representative western blots from three independent experiments (n = 3). ( D ) viral genomic and anti-genomic RNA levels form infected and treated cell lysates were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Two different primer sets were used for each RNA species—primers for N and L ORFs were used to measure genomic RNA levels, primers for the regions spanning between N and P ORFs or F and G ORFs were used to measure the levels of the anti-genomic RNA. The fold change of viral genomic or anti-genomic RNA levels normalized to microtubules mRNA levels in GA treated compared to DMSO treated are plotted. The data are mean ± SEM from three independent experiments (n = 3). ( E ) Virions from culture supernatants of (C) were isolated by pelleting through 25% sucrose cushions and hRSV Nucleocapsid expression levels were assessed as in (C). Shown is a representative western blot from three independent experiments (n = 3). ( F ) Virions from culture supernatants of (D) were isolated by pelleting through 25% sucrose cushions and qRT-PCR using two primer sets (amplifying in N and L ORFs) was used to measure the levels of genomic RNA in each sample. The fold change of genomic RNA levels in GA treated compared to DMSO treated cultures are plotted. The data are mean ± SEM from three independent experiments (n = 3).

Article Snippet: Human respiratory syncytial virus (hRSV) strain A2 nucleoprotein antibody was purchased from Sino Biologicals (Cat# 40821-T46).

Techniques: Virus, Infection, Concentration Assay, Fluorescence, Microscopy, Expressing, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Isolation

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Antiviral effects of PC786. (A) Chemical structure of PC786. (B) Western blotting image demonstrating the inhibitory effects of PC786 on RSV protein expression in HEp-2 cells infected with RSV A2 for 3 days. *, nonspecific band; F1, disulfide-linked F1 subunit cleaved from RSV F0 protein precursor. NT, no PC786 treatment (0.5% DMSO only). (C) Inhibitory effects of RSV A Memphis 37- and RSV B strain 1262-induced CPE in HEp-2 cells. (D and E) PCR viral loads in the supernatants from HEp-2 cells infected with Memphis 37 (D) and strain 1262 (E) in the presence of PC786. The assay was conducted in duplicate, and individual dots show data from individual wells.

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques: Western Blot, Expressing, Infection

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Comparison of in vitro anti-RSV activity of PC786 with those of known antiviral agents ^a

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques: In Vitro, Activity Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Anti-RSV effects of PC786 against low-passage RSV clinical isolates in HEp-2 cells, evaluated in a CPE assay and by reverse transcription-PCR

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Kinetics of antiviral activity of PC786. (A) Antiviral activity of PC786 evaluated in a time-of-addition assay. PC786 was added as a single drug exposure at 0 (just after virus inoculation), 24, 48, or 72 h after infection with RSV A2, and the resulting CPE was evaluated at 144 h postinfection, showing persistence of action. (B) GS5806 data. (C to E) PC786 (C), AZ-27 (D), or ribavirin (E) was used to dose BEAS-2B cells just after virus infection (called “nonwashout”) or added for 2 h and then removed and incubated for a further 24 h before inoculation (called “washout”). Antiviral activity was assessed by detection of RSV F protein on the cell surface using enzyme-linked immunoassay. The y axis shows percent inhibition (mean ± SEM) versus signals from vehicle-treated virus-infected controls. (F) Time course of virus titer in apical wash from air-liquid interface-cultured differentiated human bronchial epithelial cells. After infection, apical wash with PBS was collected daily up to 7 days. After each apical wash collection, PC786 was administered for 1 h apically and then removed. The limit of quantification (LOQ) is 1.5 log PFU/ml, and virus titer lower than LOQ is shown as <1.5.

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques: Activity Assay, Infection, Incubation, Inhibition, Cell Culture

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Effects of PC786 administered either intratracheally or intranasally on RSV load (PFU) in the lungs of RSV A2-infected mice

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor

doi: 10.1128/AAC.00737-17

Figure Lengend Snippet: Mode of action of PC786. (A) Inhibition of PC786 on RSV RdRp activity in crude extracts from RSV L protein-P overexpressed in HEp-2 cells. (B) Inhibition of reporter fluorescent signal in RSV RdRp-driven minigenome assay. (C) Inhibitory activity of PC786 on CPE induced by control RSV A2 virus and by a PC786 escape mutant (Y1631H). (D) SNP genotype PCR analysis of viruses with wild-type and Y1631H mutant L protein. (E) SNP genotype PCR analysis of viruses with wild-type and V153A mutant M protein. (F) Minigenome analysis in HEp-2 cells transfected with wild-type and Y1631H mutant L protein plasmid.

Article Snippet: RSV A2 large polymerase (L) helper plasmid (NR-36461), RSV A2 matrix 2-1 (M2-1) helper plasmid (NR-36464), RSV A2 nucleoprotein (N) helper plasmid (NR-36462), and RSV A2 phosphoprotein (P) helper plasmid (NR-36463) were obtained through BEI Resources, NIAID, NIH (Manassas, VA).

Techniques: Inhibition, Activity Assay, Mutagenesis, Transfection, Plasmid Preparation