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