Sera were analysed by western blotting using BTV-infected cell-lysate antigens, as previously

described [29], [30] and [32]. Anti-VP2, anti-VP5 or anti-VP7 antibodies were diluted at 1/50, while anti-mouse peroxidase-conjugated antibody was diluted at 1/750. Supernatant of BTV-4-infected BHK-21 cells was clarified by centrifugation at 3000 × g, then learn more inactivated at 56 °C for 1 h. The inactivated BTV-4 virus suspension was mixed volume to volume with 100 mM sodium carbonate buffer pH 9.6 and 100 μl was used to coat 96 well plates (4 °C for 16 h). Sera were diluted 1/100 in 5% skim-milk and ELISA were conducted as previously described [29], [30] and [32]. A serum sample from Balb/c mice immunised see more with Zulvac-4®-Bovis (inactivated BTV-4, Zoetis) was identified as the ‘standard’ against which all OD readings were subsequently normalised. Normalised optical density (NOD) was calculated as NOD = [OD (sample) − OD (Blank reaction)]/[OD (standard) − OD (Blank reaction)]. An ELISA based on clarified supernatants from non-infected cells was also used. BSR cells were grown on coverslips in 24-well plates, transfected with pCIneo-BTV-4VP2, pCIneo-BTV-4VP5, or pCIneo-BTV-4VP7 and processed for immunofluorescence as previously described [22]. Cells were probed with anti-VP2, anti-VP5 or anti-VP7 antibodies diluted 1/500 in phosphate-buffered saline containing 0.5% bovine serum albumin. BSR cells were plated

(1 × 105 cells/well) in 48 well plates a day before PRNT initiated [33]. 50 pfu of BTV-4 or BTV-8, in 125 μl of Eagle’s minimum essential medium (EMEM), were incubated with 125 μl of two-fold serial dilutions of mouse sera in EMEM, incubated at 37 °C for 2 h, then added to confluent BSR cell-monolayers. The supernatant from was discarded and replaced with molten 1% low melting point agarose (Sigma) in EMEM. Plates were subsequently incubated at 37 °C for 5 days, fixed by addition of 2 ml of 10% formaldehyde in phosphate-buffered saline per well. After removal of agarose

plugs, monoloayers were stained with 0.1% naphthalene-black solution, then washed with deionised water and plaques counted. For plaque assay, the number of plaque-forming units (PFU) was determined using the same approach, while omitting the use of mouse serum. BTV-4(SPA2003/01) infected BHK-21 cells were harvested at day 4 post-infection. Cells were centrifuged at 2000 × g and pellets were extracted with ‘RNA Now’ (Biogentex) [34]. Blood from challenged IFNAR−/− mice was extracted using ‘RNA Now’ as previously described [35] and [36]. This extraction method results in high sensitivity for viral RNA detection in mouse blood [36]. Supernatants from BTV-4 or BTV-8 infected cell-cultures were clarified at 2000 × g, concentrated 10-fold using Vivaspin® concentrators (MWCO 100K) then treated with RNase-A and benzonase to remove non-encapsidated nucleic acids.