gitrl-rpe  (Verlag GmbH)

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Characteristics of rRABV expressing GitrL in vitro. ( A ) Schematic diagrams for the construction of rLBNSE and rLBNSE-GitrL. The vector pLBNSE was derived from the SAD-B19 strain with pseudogene deletion. BsiW I and Nhe I were introduced between the G and L genes. N , P , M , G , and L represent RABV nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes, respectively. ( B ) Amino acid sequence alignment of the GitrL gene, the red area indicates the amino acid sequence was consistent with the reference sequence of the GitrL gene. ( C ) Viral titers from the fourth passage to the tenth passage on BSR cells. ( D , E ) The virus titers of different rRABVs-infected cells with MOI = 0.01 and MOI = 5 were determined at 1, 2, 3, 4, and 5 dpi, respectively. On this basis, the multi-step growth curves ( D ) and one-step growth curves ( E ) of rLBNSE- and rLBNSE-GitrL-infected BSR cells were plotted. ( F , G ) Morphology and size of viral fluorescent spots formed by different rRABVs in BSR cells. ( H ) The expression level of GitrL in the supernatant of infected cells was detected by ELISA. ( I ) Cell viability of different rRABVs-infected cells with MOI = 0.01 at 1, 2, 3, and 4 dpi, respectively. The error bar represented the mean ± standard deviation (SD, n = 3). The following symbols are used to indicate significant differences between groups: ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Plasmid Preparation, Derivative Assay, Sequencing, Virus, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: The pathogenicity of rRABV expressing GitrL. ( A ) Body weight changes in female 6-week-old ICR mice (n = 7) infected with 5 × 10 6 FFU of rLBNSE, rLBNSE-GitrL, or DMEM via i.c. pathways. ( B ) Survival rate of 5-day-old mice (n = 18) i.c. injected with 100 FFU of the above rRABVs. ( C ) N mRNA levels, ( D ) vRNA levels, ( E ) GitrL mRNA levels in the infected BALB/c mouse brain (n = 6) were quantified using RT-qPCR. The error bar indicates the mean ± standard error of the mean (SEM). The following symbols are used to indicate significant differences between groups: *, p < 0.05; ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: rLBNSE-GitrL promotes activation of DCs in vitro. ( A ) Isolation and cultivation of BMDCs. ( B ) DCs were cultured and stimulated with DMEM as the control group. ( C ) DCs were cultured and stimulated with rLBNSE. ( D ) DCs were cultured and stimulated with rLBNSE-GitrL. The white arrow indicated clustered DCs. ( E ) Gating strategies for the detection of the mature CD11C + CD80 + BMDCs, CD11C + CD86 + BMDCs, and CD11C + MHCII + BMDCs. ( F ) Representative flow cytometry plots of activated DCs. ( G ) Percentage of activated DCs (CD11C + CD80 + ) per 10 5 BMDCs after 24 h of incubation. ( H ) Percentage of activated DCs (CD11C + CD86 + ) per 10 5 bone BMDCs after 24 h of incubation. ( I ) Percentage of activated DCs (CD11C + MHCII + ) per 10 5 BMDCs after 24 h of incubation. ( J ) The expression level of IFN-α in the supernatant of activated DCs was detected by ELISA. ( K ) The expression level of IL-12p40 in the supernatant of activated DCs was detected by ELISA. The error bar represents the mean ± SD (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Activation Assay, In Vitro, Isolation, Cell Culture, Control, Flow Cytometry, Incubation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: rLBNSE-GitrL increases the number of activated DCs in vivo. ( A ) Cell populations and gating strategies for DCs by flow cytometry within LNs. ( B ) Representative flow cytometry plots of activated DCs. ( C ) Total number of activated DCs (CD11C + CD80 + ) per 10 5 draining LNs cells at 3 and 6 dpi. ( D ) Total number of activated DCs (CD11C + CD86 + ) per 10 5 draining LNs cells at 3 and 6 dpi. ( E ) Total number of activated DCs (CD11C + MHCII + ) per 10 5 draining LNs cells at 3 and 6 dpi. The error bar indicates the mean ± SEM (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: In Vivo, Flow Cytometry

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Transcriptome sequencing and differentially expressed gene analysis. ( A ) Comparative bar chart of differential genes. ( B ) Volcano plot of the differentially expressed genes between immunized mice of rLBNSE and rLBNSE-GitrL. ( C ) Gene expression heatmap. ( D ) Validation of the CXCL9. ( E ) Validation of the IL-6. ( F ) Validation of the IL-17d. ( G ) Validation of the MDP1. ( H ) Validation of the MMP2. ( I ) Validation of the STAT1. ( J ) Validation of the TIMP1. ( K ) Validation of the TNF-α. ( L ) Bar chart of KEGG enrichment pathway of the differentially expressed genes between rLBNSE and rLBNSE-GitrL. ( M ) KEGG enrichment pathway scatter plot of the difference expression genes between rLBNSE and rLBNSE-GitrL. The error bar indicates the mean ± SEM (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Sequencing, Gene Expression, Biomarker Discovery, Expressing

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Overexpression of GitrL improves antibody response and protection against pathogenic RABV challenge. ICR mice (n = 10) were i.m. vaccinated with 100 µL volume containing 10 6 FFU rLBNSE or rLBNSE-GitrL, or an equivalent amount of DMEM (as mock). Serum samples were collected at 3 and 6 dpi, and VNA titers ( A ) and GMT ( B ) were measured by FAVN assay. ( C , D ) The OD values of RABV G-specific IgG ( C ) and IgM ( D ) in the serum of immunized mice were determined by ELISA. ( E ) At 3 dpi, groups of immunized ICR mice (n = 15) were challenged with 50 LD50 of CVS-24 via i.c. route, and then monitored for another 20 days, and survivorship was recorded. The error bar indicates the mean ± SEM. The following symbols were used to indicate significant differences between groups: **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay