Journal: Scientific Reports

Article Title: The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response

doi: 10.1038/s41598-024-80708-4

Figure Lengend Snippet: The overexpression of the bovine miRNA16a inhibited BCoV replication on the viral genome copy numbers and the viral infectivity levels. ( A ) In silico prediction of miRNA16atargeting the BCoV spike gene at two different sites. The folding energy represents the binding energy of the miRNA with the target region. ( B ) Multiple sequence alignment shows that the miRNA16a/Spike binding region (indicated in red box) is conserved among nine different BCoV/Ent and BCoV/Resp isolates. ( C ) qRT-PCR analysis demonstrating the genome viral load of BCoV in scrambled- and miRNA16a-transfected MDBK cells. ( D ) qRT-PCR analysis illustrating the genome viral load of BCoV in scrambled- and miRNA16a-transfected BEC cells. ( E ) Viral plaque assay indicating the infectivity level of the BCoV enteric isolate in scrambled- and miRNA16a-transfected MDBK cells. ( F ) Western blot analysis of BCoV-nucleocapsid (BCoV-N) and BCoV-spike (BCoV-S) in the MDBK cells transfected with scrambled or miRNA16a. ( G ) Western blot band density of the BCoV-N protein normalized to that of β-actin in the MDBK cells. ( H ) Western blot band density of the BCoV-S protein normalized to that of β-actin in the MDBK cells. ( I ) BEC cells were transfected with miRNA-Scr and miRNA16a, and western blot analysis was used to assess the protein expression of BCoV-N and BCoV-S. ( J ) Western blot band density of BCoV-N protein normalized to that of β-actin in BEC cells. ( K ) Western blot band density of BCoV-S protein normalized to that of β-actin in BEC cells. All the experiments were performed in triplicate. The significance of the data was determined by one-way ANOVA with Dunnett’s multiple comparison test and indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: The primary antibodies were used to detect the expression levels of the BCoV-nucleocapsid mouse anti-bovine monoclonal (clone: FIPV3-70; Cat. No. MA1-82,189), BCoV-spike rabbit anti-bovine polyclonal (cat. no. PA5-117562), and β-actin rabbit anti-bovine polyclonal (Catalogue number: PA1-46296) antibodies were purchased from Invitrogen.

Techniques: Over Expression, Infection, In Silico, Binding Assay, Sequencing, Quantitative RT-PCR, Transfection, Viral Plaque Assay, Western Blot, Expressing, Comparison