Over 7000 samples were collected from 15 provinces of China, including Guangdong, Guangxi, Hebei, Shandong, Liaoning, Shaanxi, Hunan, Hubei, Sichuan, Jiangxi, Yunnan, Fujian, Henan, Chongqing and Jilin provinces. A total of 85 influenza virus subtype H7N9 strains were identified. Among them, 70 strains were isolated in 2017, 7 were isolated in 2018, and 8 were isolated in 2019. The isolation rate of goose-, duck- and chicken-derived strains was 28.2%, 18.8%, and 53%. The sequences of those isolates were deposited in GISAID.
A total of 20 influenza virus subtype H7N9 strains which have wide coverage and strong representativeness, were selected for further analysis based on isolation time, location, and source host (Supplementary Table S2). There were 16 strains with 4 inserted amino acids near the cleavage site of HA protein (PEVPKGKRTAR↓GLF and PEVPKRKRTAR↓GLF), which represent the molecular characteristics of HPAIV. Among the 20 strains, whilst the isolates from 2017 were of LPAIV, those from 2018 to 2019 all showed HPAIV characteristics (Ke et al. 2017; Zhu et al. 2017). The occurrence of T160A, G186V and Q226L amino acid mutations of the HA protein enhance the virus binding ability to sialic acid α-2, 6 galactose receptors (Dortmans et al. 2013). Based on our analysis, T160A, G186V, G186A and Q226L mutations occurred in 13, 19, 1 and 3 strain(s), respectively. In the E128 strain, three mutations (T160A, G186A and Q226L) occurred concurrently. Analysis on NA protein receptor binding site showed that R292K mutation occurred in 2 HPAIV strains isolated in 2019, suggesting that the drug-resistant mutation of this strain may pose a greater potential threat than that of other strains (Table 1). Similar to most early H7N9 strains, the absence of amino acids in the neck of NA protein may induce the virus adaptation to different host environments (Desmet et al. 2013). The absence of amino acids in the neck of NA protein at positions 69-73 in all the 20 isolates may indicate that strains exhibit an enhanced ability to infect and replicate in mammals (Castrucci and Kawaoka 1993).
Gene Site Effect Number HA (H3 number) PEIPKG↓GLF Multiple consecutive basic amino acids are a typical feature of highly pathogenic avian influenza 3 PEVPKG↓GLF 1 PEVPKRKRTAR↓GLF 14 PEVPKGKRTAR↓GLF 2 T160A Enhance the ability of the virus to bind to SAα2-6 Gal receptor 13 G186V 19 G186A 1 Q226L 3 NA (N2 number) H276Y Resistance to neuraminic acid inhibitors 20 R292K 2 Stalk deletion (69–73) Increase replication and virulence in mammals 20 M2 S31N Resistance to M2 ion channel blockers 20 PB2 A588V Increased adaptability and virulence to mice 6 E627K Enhance virus replication 20 D701N Enhance adaptability in mammals 20
Table 1. Statistics of key sites found in the 20 H7N9 strains
Prediction analysis of potential glycosylation sites in HA protein showed that, except for E656 strain, there were four potential glycosylation sites (30NGT32, 46NAT48, 249NDT251, 493NNT495/497NNT499) in all isolates from 2017 to 2018, which were similar to those from the previous four outbreaks. However, there were five potential glycosylation sites (30NGT32, 46NAT48, 141NGT143, 249NDT251 and 497NNT499), with an additional potential glycosylation site of 141NGT143 in the 2019 isolates (Supplementary Table S3). The loss of glycosylation site at position 150 of HA protein may change the virus*s ability in recognizing mammalian cell receptors (Gao et al. 2013). Prediction analysis of NA protein potential glycosylation sites revealed that, except for E656 and F692 strains, there were seven potential glycosylation sites (42NCS44, 52NTS54 (or 52NPS54, 52NIS54), 63NET65, 66NIT68, 82NLT84, 142NGT144 /142NET144 and 197NAS199) in all isolates from 2017 to 2018. Notably, with an additional potential glycosylation site of 354NNT356, there were eight potential glycosylation sites in 2019 isolates (Supplementary Table S4). NA proteins can hydrolyze receptor-specific glycoproteins on the surface of host cells to facilitate the release of virus particles. Changes in potential glycosylation sites suggest that the 2019 strains may have adapted to the host environment.
Taking A/Anhui/1/2013 as the root, the H7N9 strains, based on genetic variations in their HA and NA genes, are divided into the Yangtze River Delta branch and Pearl River Delta branch. According to the phylogenetic tree, it can be inferred that the strains isolated in 2018-2019 have evolved from the previously prevalent 2017 strains. Notably, both HA and NA gene of the 2019 strains form an independent small branch (Fig. 1, Fig. 2), indicating that the 2019 strains have evolved and become genetically distant from strains isolated in 2013-2018. In addition, all the isolates except E65 located in the Yangtze River Delta branch form an independent branch with some Guangdong avian influenza virus strains, same results revealed in HA and NA phylogenetic trees. Since the isolates were collected from different provinces (Fig. 1), a cross-regional genetic exchange may have occurred.
Figure 1. Phylogenic tree of H7N9 influenza viruses using HA gene sequences. The tree was rooted by using A/Shanghai/1/2013(H7N9), which was collected in February 2013.
The HA nucleotide substitution rate of isolates from the first and second waves was unchanged. Whilst those from the third and fourth wave showed a decline; and those from the fifth wave showed a slight increase. Strikingly, the HA nucleotide substitution rate of 2018-2019 strains increased 10 times than that of isolates from the fifth wave, indicating an acceleration of HA nucleotide substitution in influenza virus subtype H7N9 (Table 2).
The nucleotide substitution rate of the HA genes (site/year) First wave Second wave Third wave Forth wave Fifth wave Time -2013 2013 ~ 2014 2014 ~ 2015 2015–2016 2016–2017 2018–2019 Rate 5.755E-3 5.734E-3 3.807E-3 1.821E-3 1.917E-3 1.963E-2 95% 3.85–7.77 (E-3) 3.98–7.71 (E-3) 1.72–6.28 (E-3) 5.27E-5–3.67E-3 2.92E-4–3.60E-3 8.65E-3–3E-2
Table 2. HA nucleotide substitution rate
The mutation rate of the first nucleotide within a codon in the entire HA open reading frame of the first wave isolates was found to be increased by nearly 2 times compared to that of the second wave isolates. The mutation rate then remained roughly unchanged, with a slight increase or decrease in isolates from the third, fourth and fifth waves. Meanwhile, the mutation rate of the second nucleotide within a codon in the entire HA open reading frame was observed to be increased by 2 times in the second wave isolates compared to that in the first wave isolates. The mutation rate remained the same in the third wave isolates and subsequently decreased in isolates from the fourth and fifth waves. For the third nucleotide within a codon in the entire HA open reading frame, the mutation rate was found to be continuously decreased in isolates from the first, second and third waves; but slightly increased in isolates from the fourth wave, and then remained the same in isolates from the fifth wave (Table 3).
Codon nucleotide mutation rate First wave Second wave Third wave Forth wave Fifth wave -2013 2013–2014 2014–2015 2015–2016 2016-2017 2018 ~ 2019 First nucleotide 0.354 0.614 0.71 0.603 0.695 0.939 Second nucleotide 0.248 0.575 0.574 0.413 0.379 0.518 Third nucleotide 2.398 1.811 1.716 1.984 1.926 1.544
Table 3. Codon nucleotide mutation rate
In the reaction of H7-Re1 standard positive sera with the virus antigens of isolates, the HI titer of 2017 isolates was observed to be 4log2 ~ 8log2. Whilst isolate F11 showed the lowest HI titer of 4log2; the remaining 2017 isolates had HI titers of 5log2 ~ 8log2. The HI titers of 2018 isolates were found to be 4log2 ~ 5log2; while those of 2019 isolates were observed to be 3log2 ~ 5log2. Meanwhile, H7-Re1 standard antigen and standard positive serum showed a HI titer of 9log2. The HI titer of 2018 ~ 2019 isolates was observed to be 6log2 ~ 9log2 with H7-Re2, and 2019 isolates showed a significant antigenic difference. H7-Re3 and rLN79 had good antigenic matching with the epidemic strains in 2019 (Table 4).
Antigen Antiserum (log2) H7-Re1 H7-Re2 H7-Re3 rLN79 H7-Re1 9 12 9 10 H7-Re2 10 11 8 8 H7-Re3 3 6 10 10 rLN79 4 6 10 10 E65(2017) 7 8 9 9 E112(2017) 8 8 9 9 E122(2017) 5 6 8 9 E128(2017) 8 8 9 10 E194(2017) 5 9 8 10 E282(2017) 6 6 9 10 E594(2017) 5 6 8 10 E656(2017) 5 6 10 9 E664(2017) 6 8 9 9 E743(2017) 6 8 9 10 F11(2017) 4 9 9 10 F450(2018) 4 8 9 9 F690(2018) 5 9 10 10 F691(2018) 5 9 10 9 F692(2018) 5 9 10 10 G285(2019) 4 9 10 10 G323(2019) 5 6 9 10 G375(2019) 4 6 9 9 G502(2019) 3 6 10 10 G503(2019) 3 6 9 10
Table 4. Hemagglutination inhibition endpoint titers of 20 influenza virus subtype H7N9 strains