H7N9 virulent mutants detected in chickens in China pose an increased threat to humans

Abstract

Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin (HA) cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.

Figure 1

Genetic relationships among the HA genes and genotype evolution of H7N9 influenza viruses. (A) Phylogenetic tree of HA. The tree was rooted to A/chicken/Rostock/45/1934(H7N1) virus and based on an alignment of HA0 (nucleotides 29-1 723 of the viruses with stars and nucleotides 29-1 711 of the others). Sequences of viruses with names in black were downloaded from available databases; viruses with names in colors were sequenced in this study. The scale bar indicates the number of nucleotide substitutions per site. (B) Genotypes of the H7N9 viruses. The eight gene segments are indicated at the top of each bar. The colors of the bars represent the groups in the trees of Figure 1A and Supplementary information, Figure S1. Viruses labeled with red star(s) contain the four-amino acid (-KRTA-) insertion in their HA genes.

Figure 2

Replication and virulence of H7N9 viruses in chickens and mice. Death (A) and viral titers in organs (B) of chickens inoculated with CK/SD008. Viral titers in mice inoculated with CK/SD008, CK/SD008-PB2/627K, or CK/SD008-PB2/701N (C). Body weight change (D-F) and death (G-I) of mice inoculated with CK/SD008 (D, G), CK/SD008-PB2/627K (E, H), or CK/SD008-PB2/701N (F, I). The values or viral titers in mice were statistically analyzed by using a one-tailed paired t-test. ^aP < 0.01 compared with the corresponding value for the CK/SD008-inoculated group. The dashed lines indicate the lower limit of detection.

Figure 3

Replication and respiratory droplet transmission of H7N9 viruses in ferrets. Virus replication: (A) CK/SD008; (B) CK/SD008-PB2/627K; (C) CK/SD008-PB2/701N. Virus respiratory droplet transmission: (D) CK/S1053; (E) CK/SD008; (F) CK/SD008-PB2/627K; (G) CK/SD008-PB2/701N; (H) AH/1. Body weight change: (I) CK/S1053; (J) CK/SD008; (K) CK/SD008-PB2/627K; (L) CK/SD008-PB2/701N; (M) AH/1. Each bar (A-H) represents the virus titers from an individual animal, and the dashed red lines indicate the lower limit of detection. The red stars on F, K, and L indicate the day the animal died.

Figure 4

Receptor-binding properties of influenza viruses. Viruses were compared for their ability to bind to sialyglycopolymers containing either α2,6-siaylglycopolymer or α2,3-siaylglycopolymer.