PMC

Matrix real-time PCR and HA subtyping results for paired samples (P, V) from 32 birds before and after inoculation in ECE. HA subtypes in the original VTM samples were determined by 640PCR/Sanger sequencing, while subtypes in allantoic fluid (ALF) were determined by both 640PCR and M-RTPCR/NGS. NT = not tested. Neg = matrix Ct >45 or HA subtype not detected.

Multi-segment RT-PCR results for allantoic fluid (ALF) samples that were positive by matrix real-time PCR. Full genome sequences were determined by next generation sequencing (NGS) for samples even if they lacked detectable bands. ^a Agarose gel profiles were examined for the presence/absence of individual segment bands. If all segment bands were absent on the gel, the sample was not put through NGS. Absent bands are highlighted. P = band present, A = band absent, NT = not tested by NGS and reported as negative in . ^b The PB1 and PB2 segments are the same size and therefore cannot be resolved on agarose gels. ^c Sample 2578V was found to have two NS sequences so was designated “H3N8 mix.”