Plaque purification
Plaque assay was used to obtain purified virus clones for selected samples showing unequivocal results with pathotype-specific RT-qPCRs. Briefly, different virus dilutions were incubated on confluent LMH cell monolayers cultured in six-well plates overnight at 37°C. Then, virus inoculum was removed, cell layers washed carefully twice with sterile PBS and overlaid with modified Eagle’s medium containing 1.8 % agar. Infected cells were incubated at 37 °C and 55% humidity for three days. Finally, selected plaques were picked and propagated on LMH cells as described above for further characterization.