Lb 960 xs3

Samples were analysed for the presence of anti-EqPV-H-VP1 antibodies using the previously described luciferase immunoprecipitation system (LIPS) [19 (link),20 (link),21 (link)]. The EqPV-H-LIPS antigen VP1 was produced as described by Divers et al. [15 (link)]. Following the LIPS assay, relative light units (RLU) were determined using a plate luminometer (LB 960 XS3; Berthold, Bad Wildbad, Germany). To calculate sensitivity the mean RLU plus three standard deviations (SD) of an EqPV-H negative horse serum was defined as a cut-off limit. A potential cross-reactivity between the LIPS and other related parvoviruses could not be excluded.

Samples were analyzed regarding the presence of anti-EqPV-H-VP1 antibodies using the luciferase immunoprecipitation system (LIPS) as described by Burbelo et al. [15 (link),16 (link)] and Pfaender et al. [17 (link)]. For the EqPV-H-LIPS, the antigen VP1 was produced as described by Divers et al. [5 (link)]. Relative light units (RLU) were measured in a plate luminometer (LB 960 XS3; Berthold, Bad Wildbad, Germany). For calculation of sensitivity, a cut-off limit, analogous to Burbelo et al. (2012) and Pfaender et al. (2015), was determined and defined as the mean RLU plus 3 standard deviations (SD) of a EqPV-H negative horse serum. A cross-reaction of the LIPS with other related parvoviruses cannot be excluded.

Anti-EqPV-H-VP1 antibodies in samples were measured using the previously described LIPS assay [8 (link),23 (link)]. EqPV-H VP1 antigen was produced and used in the LIPS. Relative light units (RLU) were determined using a plate luminometer (LB 960 XS3; Berthold, Bad Wildbad, Germany). Samples were tested in technical duplicates. To calculate sensitivity, the mean RLU plus three standard deviations (SD) of an EqPV-H negative horse serum was defined as threshold. A commercial horse serum, which was previously tested positive for the presence of EqPV-H DNA, was used as positive control in all assays.

Dual-luciferase reporter assay was applied to confirm the direct binding between lnc-HOXB8-1:2 and hsa-miR-6825-5p and to explore whether CXCR3 was the direct target of hsa-miR-6825-5p. The sequence of lnc-HOXB8-1:2 and the 3’UTR sequence of CXCR3 containing the wild-type or mutant binding sites with hsa-miR-6825-5p (Supplementary Table S5) were cloned into the psiCHECK-2 vector including Firefly luciferase gene (Fluc) and Renilla luciferase gene (Rluc), respectively. lnc-HOXB8-1:2 vectors and CXCR3 vectors were cotransfected with NC or has-miR-6825-5p mimics into 293 T cells. The relative values of Fluc and Rluc were measured by Centro LB960 XS3 (Berthold, German) using Dual-Luciferase Reporter Assay Systems.

Serum samples were frozen at −80 °C after collection, prior to further processing at the Department of Molecular and Medical Virology, Ruhr University Bochum, Germany, for LIPS analysis. Serum samples were analysed in duplicate for the presence of anti-EqHV NS3-specific IgG, using the LIPS assay as described previously [7 (link),23 (link)]. Relative light units (RLU) were measured with a plate luminometer (LB 960 XS3; Berthold, Bad Wildbad, Germany). The threshold value, above which samples were regarded as NS3 IgG-positive, was calculated for each plate by using the mean value plus three standard deviations (SDs) of an EqHV-negative horse serum sample.