Emergence and Adaptation of a Novel Highly Pathogenic H7N9 Influenza Virus in Birds and Humans from a 2013 Human-Infecting Low-Pathogenic Ancestor

Abstract

Since its emergence in 2013, the H7N9 low-pathogenic avian influenza virus (LPAIV) has been circulating in domestic poultry in China, causing five waves of human infections. A novel H7N9 highly pathogenic avian influenza virus (HPAIV) variant possessing multiple basic amino acids at the cleavage site of the hemagglutinin (HA) protein was first reported in two cases of human infection in January 2017. More seriously, those novel H7N9 HPAIV variants have been transmitted and caused outbreaks on poultry farms in eight provinces in China. Herein, we demonstrate the presence of three different amino acid motifs at the cleavage sites of these HPAIV variants which were isolated from chickens and humans and likely evolved from the preexisting LPAIVs. Animal experiments showed that these novel H7N9 HPAIV variants are both highly pathogenic in chickens and lethal to mice. Notably, human-origin viruses were more pathogenic in mice than avian viruses, and the mutations in the PB2 gene associated with adaptation to mammals (E627K, A588V, and D701N) were identified by next-generation sequencing (NGS) and Sanger sequencing of the isolates from infected mice. No polymorphisms in the key amino acid substitutions of PB2 and HA in isolates from infected chicken lungs were detected by NGS. In sum, these results highlight the high degree of pathogenicity and the valid transmissibility of this new H7N9 variant in chickens and the quick adaptation of this new H7N9 variant to mammals, so the risk should be evaluated and more attention should be paid to this variant.IMPORTANCE Due to the recent increased numbers of zoonotic infections in poultry and persistent human infections in China, influenza A(H7N9) virus has remained a public health threat. Most of the influenza A(H7N9) viruses reported previously have been of low pathogenicity. Now, these novel H7N9 HPAIV variants have caused human infections in three provinces and outbreaks on poultry farms in eight provinces in China. We analyzed the molecular features and compared the relative characteristics of one H7N9 LPAIV and two H7N9 HPAIVs isolated from chickens and two human-origin H7N9 HPAIVs in chicken and mouse models. We found that all HPAIVs both are highly pathogenic and have valid transmissibility in chickens. Strikingly, the human-origin viruses were more highly pathogenic than the avian-origin viruses in mice, and dynamic mutations were confirmed by NGS and Sanger sequencing. Our findings offer important insight into the origin, adaptation, pathogenicity, and transmissibility of these viruses to both poultry and mammals.

Keywords: adaptation; emergence; highly pathogenic H7N9 influenza virus; multiple basic amino acids.

FIG 1

Representative variants of H7N9 AIVs from Guangdong Province, China. (A) Alignment of the HA cleavage site from the five waves of H7N9 infection. Position numbers are shown according to the sequence of the highly pathogenic H7N9 variant A/Chicken/Heyuan/16876/2016. Wave numbers are marked wave I to wave V. (B) Frequency of isolation of H7N9 viruses from chickens and ducks recovered from 2013 to 2017 in Guangdong Province. The time ranges of the five waves are shown.

FIG 2

Evolutionary history of H7N9 AIV. (A) Phylogenetic tree of the H7N9 HA gene. Viruses isolated from different provinces and waves are distinguished by colors. Human strains are also highlighted. All branch lengths are scaled according to the number of substitutions (subs) per site. (B) HA gene tree revealing a single cluster of highly pathogenic H7N9 variants isolated from Guangdong Province. Cleavage sites are shown at the nodes. I, a mutation (T to I) in the cleavage site of the strain A/Guangdong/Th005/2017. (C) NA gene tree of the same cluster of highly pathogenic variants for which the results are shown in panel B. (D) A map showing the region of southern China and the major national roads.

FIG 3

Phylogenetic tree of the PB2 gene sequences. The scale bar represents the number of nucleotide substitutions per site. The highly pathogenic H7N9 variants reported in the present study are marked in red, and the remaining H7N9 viruses are marked in blue. The H9N2 AIVs are marked in black. The branches are colored according to the date on which the virus was isolated.

FIG 4

Challenge of chickens and mice with five H7N9 viruses. The five challenge viruses were A/Chicken/Guangdong/SW154/2015(H7N9), A/Chicken/Heyuan/16876/2016(H7N9), A/Chicken/Huizhou/HZ-3/2016(H7N9), A/Guangdong/Th005/2017(H7N9), and A/Guangdong/Th008/2017(H7N9). (A) Mortality of inoculated chickens. Groups of 10 6-week-old SPF chickens were inoculated intranasally with 10⁵ EID₅₀/200 μl of each virus. (B) Virus titers in the lungs and brains of the first three dead inoculated chickens. (C) Mortality of groups of eight 6-week-old SPF chickens in direct contact with infected chickens. (D) Virus titers in the lungs and brains of the first three dead chickens, infected chickens euthanized at 3 dpi, and naive chickens in direct contact with infected chickens at 4 dpi. (E) Mouse mortality. Four- to 5-week-old female BALB/c mice (13 mice/group) were inoculated intranasally with 10⁶ EID₅₀ of each virus. (F) Virus titers in the lungs and brains of mice at necropsy at 4 dpi. (G) Weight changes in the mice.