Journal: Science Advances

Article Title: Transition of pseudorabies virus from latency to reactivation state selectively triggered by pathogenic bacteria

doi: 10.1126/sciadv.adw4206

Figure Lengend Snippet: ( A ) Succinct overview of PRV quiescent infection in N2a cells. ( B ) Fluorescence microscopy images depicting N2a cells infected with different doses of HuBXY/2018-mCherry with or without ACV and IFN-α treatments. The images were acquired on 8 dpi. ( C ) Fluorescence intensity of N2a cells infected with different doses of HuBXY/2018-mCherry with or without ACV and IFN-α treatments. ( D ) LAT transcript levels in N2a cells after establishing quiescent infection with HuBXY/2018-mCherry at 0.01 and 0.005 MOI. ( E ) Relative mRNA transcription levels of the early genes IE180 and the LAT in N2a cells after establishing quiescent infection with HuBXY/2018-mCherry at 0.01 MOI (694× indicates that LAT transcription levels are approximately 694-fold higher than those of IE180 transcription). These levels were normalized to the internal reference gene 28 S RNA and calculated using the 2 -ΔΔCt method (Livak method). ( F to I ) Fluorescence microscopy images obtained from HuBXY/2018-mCherry quiescent cells infected with G. parasuis –CF7066, G. parasuis –Nagasaki, and G. parasuis –SH0165 (F); S. suis –SC19, S. suis –P1/7, and S. suis –43 (G); ETEC- C83549 , ETEC-E66, and ETEC-10667 (H); and ExPEC-RS218, ExPEC-PCN033, and medium control (I). Scale bars, 100 μm. All data were analyzed using t test; *** P < 0.001 indicates significant differences.

Article Snippet: Subsequently, the infective medium was removed, and the infected cells were cultured in a medium containing ACV (MedChemExpress, NJ, USA) and IFN-α2a human recombinant (IFN-α, PROSPEC, Rehovot, Israel) for 8 days.

Techniques: Infection, Fluorescence, Microscopy, Control

Journal: Science Advances

Article Title: Transition of pseudorabies virus from latency to reactivation state selectively triggered by pathogenic bacteria

doi: 10.1126/sciadv.adw4206

Figure Lengend Snippet: ( A ) Differentially expressed genes in N2a cells upon ExPEC-induced reactivation of quiescent HuBXY/2018-mCherry. ( B ) Read counts and qPCR validation of CXCL1 transcriptional alteration. ( C ) mRNA transcriptional changes of CXCL1 in N2a cells during ExPEC-induced HuBXY/2018-mCherry reactivation. Data were analyzed using the 2 −ΔΔ C t method. ( D ) CXCL1 transcript levels in each group at 6 and 96 hpi. Data were analyzed using the 2 −ΔΔ C t method. ( E ) Differential transcription of IE180 and LAT in N2a-WT and CXCL1 -KO cells during establishment of HuBXY/2018-mCherry quiescence. ( F ) Fluorescence microscopy and bright-field imaging confirmed ExPEC-induced reactivation of quiescent HuBXY/2018-mCherry in N2a-WT cells, but no reactivation was observed in CXCL1 -KO cells. mCherry fluorescence indicates reactivated HuBXY/2018-mCherry within cells. Scale bar, 100 μm. ( G ) Quantification of HuBXY/2018-mCherry reactivation in N2a-WT and CXCL1 -KO cells by gE copy number ( n = 6). ( H ) Proliferation of HuBXY/2018-mCherry during quiescent infection (ACV + IFN-α) in N2a-WT and CXCL1 -KO cells, assessed by qPCR of gE genome copies. Infected cells were sampled from 0 to 11 dpi. Analyses were performed using t-test (** P < 0.01; *** P < 0.001; ns, P > 0.05). ( I ) Proliferation of HuBXY/2018-mCherry during natural infection in N2a-WT and CXCL1 -KO cells. Samples were collected from 0 to 11 dpi. Analyses were performed using t test (*** P < 0.001; ns, P > 0.05). ( J ) Effect of pEGFP-CXCL1 and pEGFP-vector on HuBXY/2018-mCherry reactivation in CXCL1 -KO cells. Scale bar, 100 μm. ( K ) Quantification of HuBXY/2018-mCherry gE copies in CXCL1 -KO cells transfected with pEGFP-CXCL1 or pEGFP-vector. Analyses were performed using t test (*** P < 0.001; ns, P > 0.05).

Article Snippet: Subsequently, the infective medium was removed, and the infected cells were cultured in a medium containing ACV (MedChemExpress, NJ, USA) and IFN-α2a human recombinant (IFN-α, PROSPEC, Rehovot, Israel) for 8 days.

Techniques: Biomarker Discovery, Fluorescence, Microscopy, Imaging, Infection, Plasmid Preparation, Transfection

Journal: Virology Journal

Article Title: HSV-1 immune escapes in microglia by down-regulating GM130 to inhibit TLR3-mediated innate immune responses

doi: 10.1186/s12985-024-02492-x

Figure Lengend Snippet: Effect of berberine (BBR) combined with acyclovir (ACV) on GM130 on TLR3 and inflammatory cytokines. a Cellular activity of cells treated with different concentrations of BBR for 24 h. Selection of BBR at a concentration of 3 μM pretreated for 12 h followed by HSV-1 infection for 12 h, and collection of samples. Cells are pretreated with ACV (3 μM) for 12 h and infected with HSV-1 for 12 h. b Protein levels of GM130, TLR3, and HSV-1 treated with BBR and ACV were adjusted with βactin as the internal reference. The expression levels of the detected proteins in each group were compared with those of the uninfected group. c Semi-quantitative results of GM130. d Semi-quantitative results of TLR3. e Semi-quantitative results of HSV-1-gD. f – h Secretion levels of the inflammatory cytokines IFN-β, TNF-α, and IL-6 after treatment with BBR and ACV. i Viral titers are treated with BBR and ACV. Data are obtained through three independent replicates, expressed as X ± SD, and statistically analyzed between the two groups using a t-test. A comparison between the two groups is marked with a dashed line. ns, p > 0.05; *, #, p < 0.05; **, ##, p < 0.01; ***, ###, p < 0.001

Article Snippet: To observe the effects of BBR hydrochloride (Sigma, Cat#BP1108) and BBR in combination with ACV (MCE, Cat#HY-17422), we used BBR (0, 3, 10 uM), ACV (3 μM), and BBR (3 uM) + ACV (3 uM) treating BV2 cells separately at 24 h before sample collection.

Techniques: Activity Assay, Selection, Concentration Assay, Infection, Expressing, Comparison

Journal: Virology Journal

Article Title: HSV-1 immune escapes in microglia by down-regulating GM130 to inhibit TLR3-mediated innate immune responses

doi: 10.1186/s12985-024-02492-x

Figure Lengend Snippet: Structural changes of the Golgi apparatus (GA) in microglia after treatment with acyclovir (ACV) and berberine (BBR). ACV and BBR were pretreated for 12 h before infection, HSV-1 infected cells with MOI = 1, and GA structure (GM130 marker, P115 marker) is observed 12 h after infection. Scale, 10 μm. After ACV treatment, the fluorescently labeled GA structure of GM130 and P115 partially recovered compact perinuclear distribution. After BBR treatment, the fluorescently labeled GA structure of GM130 and P115 partially recovered compact perinuclear distribution. After ACV is combined with BBR, the fluorescently labeled GA structure of GM130 and P115 is further restored to a compact perinuclear distribution. The representative images are obtained from three independent repeated experiments. Scale, 10 μm

Article Snippet: To observe the effects of BBR hydrochloride (Sigma, Cat#BP1108) and BBR in combination with ACV (MCE, Cat#HY-17422), we used BBR (0, 3, 10 uM), ACV (3 μM), and BBR (3 uM) + ACV (3 uM) treating BV2 cells separately at 24 h before sample collection.

Techniques: Infection, Marker, Labeling