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).