Clade 2.3.2.1 H5N1 avian influenza viruses circulate at the interface of migratory and domestic birds around Qinghai Lake in China

During 2012-2015, six H5N1 avian influenza viruses were isolated from domestic birds and the environment around Qinghai Lake. Phylogenetic analysis of HA genes revealed that A/chicken/Gansu/XG2/2012 (CK/GS/XG2/12) belonged to clade 2.3.2.1a, while A/environment/Qinghai/1/2013 (EN/QH/1/13), A/chicken/Qinghai/QH1/2015 (CK/QH/QH1/15), A/chicken/Qinghai/QH2/2015 (CK/QH/QH2/15), A/chicken/Qinghai/QH3/2015 (CK/QH/QH3/15), and A/goose/Qinghai/QH6/2015 (GS/QH/QH6/15) belonged to clade 2.3.2.1c. Further analysis of the internal genes of the isolates found that the PB2 gene of EN/QH/1/13 had 99.6% nucleotide identity with that of A/tiger/Jiangsu/1/2013 (H5N1), which clustered into an independent branch with PB2 from multiple subtypes. PB2, PB1, and M genes of CK/QH/QH3/15 were from H9N2, suggesting it was a reassortant of H5N1 and H9N2. Animal studies of three selected viruses revealed that CK/GS/XG2/12, EN/QH/1/13, and CK/QH/QH3/15 were highly lethal to chickens, with intravenous pathogenicity indexes (IVPIs) of 2.97, 2.81, and 3.00, respectively, and systemically replicated in chickens. In a mouse study, three selected H5N1 viruses were highly pathogenic to mice and readily replicated in the lungs, nasal turbinates, kidneys, spleens, and brains. Therefore, isolates in this study appear to be novel reassortants that were circulating at the interface of wild and domestic birds around Qinghai Lake and are lethal to chickens and mice. These data suggest that more extensive surveillance should be implemented, and matched vaccines should be chosen for the domestic birds in this area.