Virus Isolation and Identification
Between 2013 and 2018, a total of 5110 environmental samples were collected from the sampling sites for poultry-related environmental surveillance in Anhui province. Analysis revealed that 582 environmental samples were positive for the H9N2 AIVs. From these samples, we obtained 33 strains using SPF chick embryonated eggs (the H9N2 isolation rate was 5.67%, Table S1). To further understand the molecular evolution of the H9N2 strains in different geographical locations, we selected all isolates from eight cities in Anhui province to construct a genetic evolutionary tree, including 16 isolates from Hefei, six isolates from Anqing, three isolates from Huaibei, two from Fuyang, two from Huainan, two from Maanshan, one from Chuzhou and one from Bengbu (Figure 1).
Table S1 . Basic information for the H9N2 avian influenza viruses isolated from the environmental samples in Anhui province from 2013 to 2018
Phylogenetic Analyses of the HA and NA genes
To better understand the genetic relationships between the H9N2 viruses in Anhui province, we sequenced the complete genomes of 33 representative strains. The genome sequences all shared high sequence similarity at the nucleotide level. For example, the HA and neuraminidase (NA) genes shared nucleotide identities of 93.2% to 96.4% and 93.3% to 99.3%, respectively.
As described in a previous report[17], four stem evolutionary clades of h9.1–h9.4 have been designated to map the HA gene phylogeny. All of the HA genes of the viruses in this study belonged to clade 9.4.2.5 represented by A/chicken/Guangxi/55/2005(H9N2), which differed from the A/quail/Hong Kong/G1/1997(H9N2)-like (G1-like, h9.4.1.1) and A/duck/Hong Kong/Y280/1997(H9N2) (Y280-like, H9.4.2.4) viruses prevailing in most Asian countries since 1994 (Figure 2A). The NA genes of all of the H9N2 viruses in this study belonged to the A/chicken/Beijing/1/1994(H9N2)-like (BJ/94-like) viruses and formed four branches together with the G1-like, G9-like and Y28-like viruses. All of the NA genes were closely clustered together with those of the Y280-like viruses, forming one group (Figure 2B).