Inhibition of clathrin-mediated endocytosis does not block BTV-1 uptake or infection.

<p>BHK cells were transfected to express GFP-Eps15 control (A–D), GFP-DN-Eps15 (E–H) or AP180C-c-myc (I–N). Cells expressing a transgene are shown in green. The transfected cells were incubated with Alexa-568 transferrin or with BTV-1 (shown as red; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011360#s2" target="_blank">Methods</a>). Panels (A), (E) and (I) show transferrin uptake, whereas panels (B), (F) and (J) show merged images for transferrin and transgene expression for the same cells as in panels A, E and I. Panels (C), (G) and (K) show uptake of BTV-1, whereas panels (D), (H) and (L) show merged images for virus labelling and transgene expression for the same cells as in panels C, G and K. Alternatively, AP180C transfected cells were infected with BTV-1 (m.o.i. = 1). At the end of the infection the cells were processed for confocal microscopy using Orab1 (anti-NS2) to identify infected cells (shown in red on Panels M and N). Panel (M) shows infected cells whereas panel (N) show a merged image for infection and AP180C expression for the same cells as in panel M. The cell nuclei are shown in blue. Scale bar  = 10 µm. Panel (O): Cells were scored positive for transferrin or BTV-1 uptake, or for infection as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011360#s2" target="_blank">methods</a>. For AP180C, both the expressing and the non-expressing cell populations were or scored for transferrin uptake (total cells counted for each population >900; <i>n</i> = 6 experiments), for BTV uptake (>500 cells; <i>n</i> = 4) and for infection (>300; <i>n</i> = 3). The level of transferrin or BTV-1 uptake, or infection by the cells expressing AP180C (EC) was normalised to the level of uptake or infection by the cells of the non-expressing population (NEC). For Eps15 experiments, cells were scored for transferrin (>280 cells; <i>n</i> = 4) or BTV-1 uptake (>280 cells; <i>n</i> = 4) and level of uptake by the cells expressing DN-Eps15 (DN) was normalised to the results for the cells expressing the Esp15 control. The mean ± SD is shown for the independent experiments (<i>P</i> values: *** <0.001).</p

Active endosomal acidification is required for BTV infection.

<p>BHK cells were mock-treated or pre-treated (PT) with 12 nM concanamycin-A, and then infected with BTV-1 (m.o.i. = 1). Alternatively, concanamycin-A was added to the cells coincident with the start of infection (Time 0 on panel C) or at the noted times after infection was initiated. At the end of the infection the cells were processed for confocal microscopy counting at least 700 cells per time point. Infected cells were identified using Orab1 (anti-NS2) and are shown in red. Panel (A) shows mock treated cells infected with BTV-1. Panel (B) shows infected cells that had been pre-treated with drug. The cell nuclei are shown in blue. Scale bar  = 10 µm. Panel (C): The level of infection of the drug treated cells was normalised to the levels of infection of the mock treated cells. The mean of duplicate samples for one experiment of two is shown, each giving similar results.</p

During entry BTV-1 is delivered directly to LAMP-1 positive compartments.

<p>BTV-1 was pre-bound to BHK cells and then internalised for 15, 30, or 120 minutes and the cells processed for confocal microscopy using PM10 to detect input virus (green). Panels A–F: Alexa-568 labelled transferrin was added to the cells during the final 15 minutes of virus uptake, to label early and re-cycling endosomes. Panels (A) and (D) show transferrin uptake. Panel (B) and (E) show virus uptake. Panel (C) and (F) show merged images for the same cells. Alternatively, cells were double labelled for BTV-1 and LAMP-1. Panels (G) and (J) shows labelling for LAMP-1. Panel (H) and (K) show virus uptake. Panel (I) and (L) show merged images for the same cells. Yellow indicate areas of co-localisation. The cell nuclei are shown as blue. Scale bar  = 10 µm.</p

Actin disruption inhibits BTV-1 infection.

<p>BHK cells were treated with 2 µM cytochalasin-D and allowed to internalise 568-Aexa labelled transferrin (shown as red) for 15 minutes (Panel C), or 568-Alexa labelled dextran (shown as red) (Panel D) or BTV-1 (shown as green) (Panels B) for 0.5 h. Panel A show virus uptake by mock-treated cells. The cell nuclei are shown in blue. Scale bar  = 10 µm. BHK cells were pre-treated (Pre) with 2 µM cytochalasin-D or 1.5 µM latrunculin-A, or mock-treated and then infected with BTV-1 (m.o.i. = 1). The drugs were also added to other cells after the start of infection (Post) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011360#s2" target="_blank">Methods</a>). At the end of the infection the cells were labelled using Orab1 (anti-NS2) and scored for infection by confocal microscopy counting at least 300 cells per condition. The level of infection of the drug-treated cells was normalised to the level of infection of the mock-treated cells. The mean ± SD for triplicate samples of one experiment representative of two is shown, each giving similar results.</p

DN-dynamin-2 does not inhibit BTV-1 infection.

<p>BHK cells were transfected to express wt dynamin-2 (Panels A, B, E and F) or DN-dynamin-2 (panels C, D, G and H). Cells expressing the transgene are shown in green. The transfected cells were incubated with Alexa-568 transferrin or with BTV-1 (shown as red). Panels (A) and (C) show transferrin uptake, whereas panels (B) and (D) show merged images for transferrin and transgene expression for the same cells as in panels A and C. Panels (E) and (G) show uptake of BTV-1, whereas panels (F) and (H) show merged images for virus and transgene expression for the same cells as in panels E and G. The cell nuclei are shown in blue. Scale bar  = 10 µm. Alternatively, transfected cells were infected with BTV-1 (m.o.i. = 1). At the end of the infection the cells were labelled using Orab1 (anti-NS2) and scored for infection by confocal microscopy. Panel (I): Cells were scored positive for transferrin or BTV-1 uptake, or for infection as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011360#s2" target="_blank">methods</a> counting at least 220 cells (<i>n</i> = 3 experiments) for each condition. The level of transferrin or BTV-1 uptake, or infection by the cells expressing DN-dynamin-2 (DN) was normalised to the level of uptake or infection by the cells expressing wt dynamin-2 (wt). The mean ± SD is shown for three independent experiments (<i>P</i> values: *** <0.001).</p

EIPA and dynasore inhibit BTV-1 endocytosis.

<p>BHK cells were mock-treated (A–C) or pre-treated with 100 µM dynasore (D–F), or 100 µM EIPA (G–I) and allowed to internalise BTV-1 (Panels A, D and G) or Alexa-568 labelled dextran (Panels C, F and I) for 0.5 h, or Alexa-568 labelled transferrin (Panels B, E and H) for 15 minutes, and processed for confocal microscopy using PM10 to detect input virus (shown as green). The cell nuclei are shown in blue. Note: EIPA treatment results in a faint blue fluorescence throughout the cytosol. Scale bar  = 10 µm.</p

BTV-1 is co-localised with dextran during cell-entry.

<p>BTV-1 was pre-bound to BHK cells and then internalised in the presence of Alexa-568 dextran (red) for 0.5 h. The cells were then processed for confocal microscopy using PM10 to detect input virus (green). Panels (A) and (B) shows dextran uptake for representative cells. Panels (C) and (D) shows virus uptake for the same cells as in panels A and B respectively. Panels (E) and (F) show merged images for the same cells. Yellow indicates co-localisation. The cell nuclei are shown in blue. Scale bar  = 10 µm.</p

Production of neutralizing anti-FMDV antibodies in cattle.

<p>Sera from the calves in group 1 (C1-C3, black bars, control), group 2 (C4-C6, red bars, vaccinated with rSFV-FMDV-P1-2A-mIRES-3C on PVD 0 followed by empty capsids on PVD 14) and group 3 (C7-C9, blue bars, vaccinated with empty capsids on PVD 0 followed by rSFV-FMDV-P1-2A-mIRES-3C on PVD 14) were collected during the experiment 3. All animals were challenged with FMDV on PVD 28. Samples from PVD 14 (A), PVD 28 (B) (prior to challenge) and PVD 36 (C) (post challenge) were assayed for the presence of neutralizing anti-FMDV antibodies using VNTs. Results were calculated as reciprocal VNT titres and are displayed as log₂ values. VNT titres ≤ 11 (log₂ 11 = 3.459) are considered negative while titres ≥45 (log₂ 45 = 5.492) are considered positive. Intermediate titres are considered as inconclusive (indicated within grey bar).</p

Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from unvaccinated and rSFV-FMDV vaccinated calves in experiment 2.

<p>Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from unvaccinated and rSFV-FMDV vaccinated calves in experiment 2.</p

Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from calves in experiment 3.

<p>Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from calves in experiment 3.</p