Genotypic analysis of the HA gene in viruses isolated from directly inoculated pigs, from contact pigs, and from ferrets after transmission.

Transmission experiments were performed as described in Table 2. Viral RNA was extracted from nasal swabs and washes collected from donor pigs (D1, D2, D3, D4, and D5), contact pigs (C1, C2, and C3) and ferrets (F1, F2, and F3). Gene-specific PCR products were obtained for the HA, NA, and M segments and analyzed by next-generation sequencing (with Illumina MiSeq). (A, B) The mean variation frequency for each sample/time point was calculated by using positions that were variable in at least one of the examined samples/reads and is displayed as the mean pairwise differences between all variants in a sample and the inoculum. One-way ANOVA analysis followed by a Tukey post-hoc test was performed to compare groups, and significant differences are shown (*P<0.05, ***P < 0.001). (C) Heat map of major amino-acid changes occurring in pigs and ferrets in the Y17H group. The results from the 2 independent experiments (transmission set-ups 1 and 2) are shown separately, and only those mutations that were transferred from the donor to a contact pig or ferret are displayed. Variants occurring with a frequency greater than 5% are reported according to H3 numbering and their position in HA1 and HA2. No mutations were found in the NA or M genes. ns, not significant; dpi, day post-inoculation.