Evolution of H7N9 highly pathogenic avian influenza virus in the context of vaccination

Abstract

Human infections with the H7N9 influenza virus have been eliminated in China through vaccination of poultry; however, the H7N9 virus has not yet been eradicated from poultry. Carefully analysis of H7N9 viruses in poultry that have sub-optimal immunity may provide a unique opportunity to witness the evolution of highly pathogenic avian influenza virus in the context of vaccination. Between January 2020 and June 2023, we isolated 16 H7N9 viruses from samples we collected during surveillance and samples that were sent to us for disease diagnosis. Genetic analysis indicated that these viruses belonged to a single genotype previously detected in poultry. Antigenic analysis indicated that 12 of the 16 viruses were antigenically close to the H7-Re4 vaccine virus that has been used since January 2022, and the other four viruses showed reduced reactivity with the vaccine. Animal studies indicated that all 16 viruses were nonlethal in mice, and four of six viruses showed reduced virulence in chickens upon intranasally inoculation. Importantly, the H7N9 viruses detected in this study exclusively bound to the avian-type receptors, having lost the capacity to bind to human-type receptors. Our study shows that vaccination slows the evolution of H7N9 virus by preventing its reassortment with other viruses and eliminates a harmful characteristic of H7N9 virus, namely its ability to bind to human-type receptors.

Keywords: Avian influenza virus; H7N9; evolution; receptor-binding properties; pathogenicity; antigenicity.

Figure 1.

Phylogenetic analyses and genotypes of H7N9 viruses. The phylogenetic trees of the HA (a) and NA (b) genes were rooted to A/chicken/Rostock/45/1934 (H7N1) and A/chicken/Italy/22A/1998 (H5N9), respectively. The H7N9 strains reported in this study are marked with black dots. The four H7N9 viruses that provided surface genes for the H7-Re1, H7-Re2, H7-Re3, and H7-Re4 vaccine seed strains are shown in red in parentheses. (c) Genotypes of H7N9 viruses and hosts in which these genotypes were detected. The numbers of strains for each genotype in each time period are provided in parentheses. The genotypes of the viruses isolated between February 2017 and December 2019 were reported previously [30,32].

Figure 2.

Replication and virulence of H7N9 viruses in mice. Virus titres in organs of mice inoculated i.n. with 10⁶ EID₅₀ of different H7N9 viruses. Three mice from each group were euthanized and their organs were collected on Day 3 p.i. for virus titration in eggs. Data shown are means ± standard deviations. The values labelled with one red star indicate that the virus was detected in the organ of only one mouse, and two red stars indicate that the virus was detected in the organ of two mice. The dashed lines indicate the lower limit of detection. The maximum body weight change in the groups of five mice after inoculation with 10⁶ EID₅₀ of the test virus are shown on the right side.

Figure 3.

Receptor-binding properties of H7N9 viruses. The receptor-binding properties of H7N9 viruses are detected with sialylglycopolymers (α−2,3-sialylglycopolymer, blue; α−2,6-sialylglycopolymer, red). The data shown are the means of three repeats; the error bars indicate standard deviations.

Figure 4.

Viral titres in organs of chickens intranasally infected with H7N9 virus. Groups of three 6-week-old SPF chickens were intranasally inoculated with 0.1 ml of inoculum containing 10⁶ EID₅₀ of the indicated virus. The chickens were euthanized on Day 3 post-inoculation and their organs were collected for viral titration in eggs.

Figure 5.

Antigenic cartography of H7N9 viruses. The antigenic map was generated using the HI assay data shown in Table S1. Each unit in the coordinates represents a 2-fold difference in the HI titre. The ovals coloured red and dark blue represent the antisera generated from the H7-Re3 and H7-Re4 vaccine strains, respectively. The ovals coloured pink and light blue show the viruses H7-Re3 and H7-Re4, respectively. The ovals coloured green represent the test viruses.

Figure 6.

Key mutations in HA1 that contribute to the antigenic difference between the H7-Re3 and H4-Re4 viruses. (a) The amino acid differences in the HA1 between the H7-Re3 and H7-Re4 vaccine strains are shown in the 3D structure of the HA1 protein. The amino acid residues located on the surface of the globular head of the HA1 protein are shown in red. (b) Cross-reactive HI antibody titres of different H7-Re3 mutants against H7-Re3 antiserum, H7-Re4 antiserum, and an H7N9 monoclonal antibody.