Results of commercially available Schmallenberg virus antibody ELISAs and of the standard microneutralization tests performed by the participating laboratories.

The sample status is given below the respective sample identifier.</p

Results of the standard microneutralization tests for the serological panel.

All results of a particular participant are depicted by the identical symbol for each sample.</p

Quantification cycle (Cq) values produced by the ring trial participants using different real-time RT-PCR systems.

Each outlier is depicted by a dot.</p

Status of the samples sent for Schmallenberg virus (SBV) infection diagnosis to the ring trial participants.

The results of the pre-testing by real-time RT-PCR [33] or microneutralization test [36] prior to shipment and the time points at which the samples for viral genome detection were taken after experimental infection are given in parenthesis. Cq–quantification cycle value, dpi–days post infection.</p

Sequences of primers and probes.

<p>FAM 5′ modification was used for IL-1β, IL-2, IL-4, IL-6, IL-8, IFN-α, TNF-α; HEX for β-Actin; Texas Red for GAPDH. BHQ (Black-Hole-Quencher)-1 was used for 3′ modifications of IL-1β, IL-2, IL-4, IL-6, IFN-α, β-Actin and GAPDH; BHQ-2 was used for of TNF-α and IL-8. Corresponding references are given in the right column. Sequences marked with “this study” were created by the use of “Primer-BLAST” available on NCBI GenBank.</p><p>F =  Forward Primer; R =  Reverse Primer; P =  Probe; bp = base pairs.</p><p>Sequences of primers and probes.</p

In-vitro cytokine stimulation.

<p>The stimulating agents are presented along with the corresponding target cytokines as well as background information about their functionality.</p><p>LPS = <i>Salmonella typhimurium</i> lipopolysaccharid; a component of the outer gram positive bacteria membrane, antigenic effect on PBMCs.</p><p>PGN =  <i>Staphylococcus aureus</i> peptidoglycan; a stabilizing macro molecule in the cell wall of gram positive bacteria; antigenic effect on PBMCs.</p><p>ConA =  Concanavalin A; a lectin from the jack bean, mitogenic effect (especially on T-cells).</p><p>PWM = Pokeweed mitogen; a lectin of the American pokeweed, activating effect on B- and T-cells.</p><p>PHA =  Phytohemagglutinin; a herbal lectin, mitogenic effect (especially on T-cells).</p><p>In-vitro cytokine stimulation.</p

Composition of the synthetic standard gene comprising all target cytokines (IL-2, IL-4, IL-8, TNF-α, IFN-α, IL-6, IL-1β) and internal reference genes (β-Actin, GAPDH, HPRT (Hypoxanthin-Guanin-Phosphoribosyltransferase), starting with the T7-promotor sequence and concluding with the NOD1 restriction site as initial point for linearization and transformation to RNA.

<p>Each target cytokine was included with a nucleotide overhang of approximately 50 base pairs (bp) prior to forward primer sequence. In total, the synthetic standard gene comprises 1464 bp.</p

Model of the receptor binding and modulating activity of the known IAV surface glycoproteins.

(A) Infection of a host cell is initiated by binding of HA subtypes H1–16 to sialic acid residues exposed on the host cell surface. These glycan structures are subsequently cleaved off by NA of the subtypes N1–9 in order to facilitate the release of viral particles. (B) The H17 and H18 HA proteins of New World bat IAVs utilize MHC-II molecules for cell entry. Preliminary data suggest that the New World bat IAV N11 NA protein decreases MHC-II surface expression by a yet unknown mechanism, allowing unhindered release of budding particles. (C) The New World bat IAV subtype H18N11 exhibits an unforeseen high flexibility to quickly acquire H18mut that compensate for an N11trunc and restore efficient growth in cell culture and mice. (D) This inherent flexibility of H18 might have the potential to allow further adaptations to new cell surface receptors. H18mut, mutations in H18; HA, hemagglutinin; IAV, influenza A virus; MHC-II, major histocompatibility complex class II; N11trunc, truncated N11; NA, neuraminidase.</p

Phylogenetic analysis of the internal gene segments of conventional and bat-derived IAVs highlights the large genetic divergence and recent reassortment events.

(A) The phylogenetic relationship of conventional and bat-derived IAVs was computed in a SuperNetwork [28] by using the nucleotide sequences of the internal gene segments (PB2, PB1, PA, NP, M, and NS) of 110 representative IAVs and six IBVs. All conventional non-bat IAVs (blue) are of common origin and cluster tightly. In contrast, the internal gene segments of the bat-derived IAVs form two outgroups that are located at a more basal position. Notably, Old World (red) and New World bat IAVs (purple) are widely separated. The parallel lines indicate uncertainties between the phylogenetic trees that make up the presented phylogenetic network. (B) A time-calibrated phylogeny was calculated for PB1 as a representative IAV internal gene segment. The timeline (presented in CE) shows that the New World bat IAV segments branched off more than 650 years ago (purple node) and that the last common ancestor of Old World bat IAVs (red node) and conventional IAVs (blue node) is around 300 years old. (C) Surprisingly, a comparable time-calibrated phylogeny of the Old World bat H9 HA (red node) along with conventional H8, H9, and H12 HAs (blue nodes) points to a much more recent common ancestor for the Old World bat–derived HA. See supporting information for a detailed description of the performed phylogenetic analysis (S1 Technical Appendix) and a full-sized version of the phylogenetic network (S1 Fig). HA, hemagglutinin; IAV, influenza A virus; IBV, influenza B virus; NP, nucleoprotein.</p

Samples from different animal trials (n = 402) used for assay validation.

<p>Samples were chosen to represent different pig species (wild boar, domestic pigs) and inoculation status (CSFV infection/vaccination, ASFV infection, corresponding control animals). Moreover, PBMC and EDTA blood samples were included.</p><p>Samples from different animal trials (n = 402) used for assay validation.</p