Serologic assays were performed as previously described (1) . The sera were all treated with receptor destroying enzyme (RDE) (Seika, Japan) and hemadsorbed on packed horse red blood cells (red blood cell). Four hemagglutination units of D/bovine/Nebraska/9-5/2012
Page 2 of 2 (kindly provided by Dr. Ben Hause) or D/bovine/France/5920/2014 and 1% horse red blood cell were used for HI assays. To check for influenza C virus putative cross-reactivity, we used C/Victoria/1/2011 (kindly provided by Dr. Richard Webby) and 1% chicken red blood cell.
The MN assays were carried out on swine testis cells (ATCC), using 100 tissue culture infectious doses 50 per well and 5 days' incubation at 37°C and 5% CO2 without TPCK trypsin. HI and MN titers were considered positive when ≥10.
Positive IDV reference serum was produced in-house by inoculating rabbits subcutaneously with purified D/bovine/Nebraska/9-2/2012. We also used IDV positive French cattle serum (from an experimental infection) and BEI Resources (NIAID) chicken antiserum C/Taylor/1233/47. The reference sera were RDE treated and hemadsorbed. When adsorbing both (i) the IDV reference antiserum on ICV and (ii) the ICV reference antiserum on IDV, and retesting for the respective antigens, we lost 1 log2 in titer, suggesting a 2-fold difference in HI titer is the "normal" loss of antibody due to the additional treatment of the sera. We therefore considered a 2 log2 decrease in titer as the threshold to consider crossreactivity between ICV and IDV.