Figure 5

Aptamer IBRV-A4 blocks entry of BOHV-1 into MDBK cells. (A) Virus titers of BoHV-1 were determined after treatment with DNA library or aptamer IBRV-A4 at 2, 4, 8, 16 and 32 hours post-infection. Controls were BoHV-1 only or mock. The statistical differences between treated and control groups were determined and analyzed using SPSS software. “**” indicated statistically significant difference (p < 0. 01). (B) Virus replication of BoHV-1 in MDBK cells was determined by real-time PCR after treatment with DNA library or aptamer IBRV-A4 at 2, 4, 8, 16 and 32 hours post-infection. Controls were either untreated MDBK cells (negative) or MDBK cells treated with a mixture of DNA library (or PBS) and BoHV-1 (positive). “***” indicated statistically significant difference (P < 0. 001). (C) Laser confocal microscopy showed that aptamer IBRV-A4 inhibited BOHV-1 infection by perturbing viral entry into MDBK cells. The Mock represented uninfected cells (a1-a3), MDBK cells infected with BOHV-1 and pre-treated with PBS (b1-b3), DNA library treated cells (c1-c3) and aptamer IBRV-A4 treated cells (d1-d3). Magnification = 200x. (D) MDBK cells infected with 100 TCID50 of BoHV-1 and treated with 0.625 nM of DNA library or 0.625 nM of aptamer IBRV-A4 at 0, 0.5, 1, 1.5, 2 and 2.5 hours post-infection. BoHV-1 titers from each time point were determined. The black line represents BoHV-1 infected MDBK cells treated with DNA library. The red dotted line represents BoHV-1 infected MDBK cells treated with aptamer IBRV-A4. “***” indicated statistically significant difference (p < 0. 001), “**” indicated statistically significant difference (p < 0. 01). (E) MDBK cells grown on coverslips and infected with 100 TCID50 of BoHV-1, then treated with 0.625 nM of DNA library or 0.625 nM of aptamer IBRV-A4 at 0, 0.5, 1, 1.5, 2 and 2.5 hours post-infection. BoHV-1 in MDBK cells was detected by IFA based on anti-BOHV-1 gD Monoclonal antibody. MDBK cell membrane was stained with DiI (1,1′-Dioctadecyl -3,3,3′,3′-tetramet hylindocarbocyanine perchlorate). Cellular nucleus was stained with DAPI. Cells were observed under laser confocal microscopy. BoHV-1 in MDBK cells is shown in green, cell membrane is shown in red and nuclei are shown in purple. Magnification = 200x.