Emergence, Evolution, and Biological Characteristics of H10N4 and H10N8 Avian Influenza Viruses in Migratory Wild Birds Detected in Eastern China in 2020

Abstract

H10Nx influenza viruses have caused increasing public concern due to their occasional infection of humans. However, the genesis and biological characteristics of H10 viruses in migratory wild birds are largely unknown. In this study, we conducted active surveillance to monitor circulation of avian influenza viruses in eastern China and isolated five H10N4 and two H10N8 viruses from migratory birds in 2020. Genetic analysis indicated that the hemagglutinin (HA) genes of the seven H10 viruses were clustered into the North American lineage and established as a novel Eurasian branch in wild birds in South Korea, Bangladesh, and China. The neuraminidase (NA) genes of the H10N4 and H10N8 viruses originated from the circulating HxN4 and H5N8 viruses in migratory birds in Eurasia. We further revealed that some of the novel H10N4 and H10N8 viruses acquired the ability to bind human-like receptors. Animal studies indicated that these H10 viruses can replicate in mice, chickens, and ducks. Importantly, we found that the H10N4 and H10N8 viruses can transmit efficiently among chickens and ducks but induce lower HA inhibition (HI) antibody titers in ducks. These findings emphasized that annual surveillance in migratory waterfowl should be strengthened to monitor the introduction of wild-bird H10N4 and H10N8 reassortants into poultry. IMPORTANCE The emerging avian influenza reassortants and mutants in birds pose an increasing threat to poultry and public health. H10 avian influenza viruses are widely prevalent in wild birds, poultry, seals, and minks and pose an increasing threat to human health. The occasional human infections with H10N8 and H10N3 viruses in China have significantly increased public concern about the potential pandemic risk posed by H10 viruses. In this study, we found that the North American H10 viruses have been successfully introduced to Asia by migratory birds and further reassorted with other subtypes to generate novel H10N4 and H10N8 viruses in eastern China. These emerging H10 reassortants have a high potential to threaten the poultry industry and human health due to their efficient replication and transmission in chickens, ducks, and mice.

Keywords: H10N4; H10N8; avian influenza virus; replication and transmission; wild birds.

FIG 1

Phylogenetic tree of the HA genes of the H10 viruses. The phylogenetic tree was constructed by MEGA 7.0 with the neighbor-joining method. The sequence names in red are the viruses isolated in this study, and the sequences in green, blue, and black were downloaded from the database.

FIG 2

Phylogenetic analysis of the NA genes of the H10N4 and H10N8 viruses. (A) NA phylogenetic tree of H10N4 viruses. (B) NA phylogenetic tree of H10N8 viruses. The sequence names in green and blue indicate the H10N8 viruses we isolated in this study, and the sequence of A/whooper swan/Shandong/SC195/2021 (H5N8) is that of the H5N8 virus isolated in our previous study. The phylogenetic tree was constructed by MEGA 7.0 with the neighbor-joining method.

FIG 3

Proposed transmission and reassortment of H10Nx viruses in wild migratory birds in North America and Eurasia. The transmission routes of the HA gene of the H10Nx viruses are indicated with arrows. Sampling points are marked with black circles. The migratory flyways are marked with different colors. Wild-bird H9N2-like viruses were isolated in the Yellow River Delta in eastern China in 2019.

FIG 4

Receptor-binding properties and sequence differences in the receptor-binding domain of HA of H10N4 and H10N8 viruses. (A to D) Receptor-binding specificity of H10N4 and H10N8 viruses. Two different glycopolymers (α-2,3-siaylglycopolymer and α-2,6-siaylglycopolymer) were used to test the receptor-binding properties of the viruses. The data shown are the means of three replicates; the error bars indicate the standard deviation. (E) Comparison of the amino acids of the receptor-binding region in HA of the representative wild bird, chicken, mink, seal, and human H10 isolates.

FIG 5

Replication, virulence, and pathological lesions of H10N4 and H10N8 isolates in mice. (A) Body weight change of the mice infected with H10N4 and H10N8 isolates. (B to E) Viral titers in mice inoculated with 10⁶ EID₅₀ of the viruses. (A) BTG/W1496/20 H10N8, (B) SW/W3875/20 H10N8, (C) SW/W4322/20 H10N4, (D) EC/W4446/20 H10N4. (F to I) Pathological lesions of the lungs of mice inoculated with the viruses. (F) BTG/W1496/20 H10N8, (G) SW/W3875/20 H10N8, (H) SW/W4322/20 H10N4, (I) EC/W4446/20 H10N4.

FIG 6

Replication of H10N4 and H10N8 isolates in chickens and ducks. Viral titers of BTG/W1496/20 H10N8, SW/W3875/20 H10N8, and EC/W4446/20 H10N4 in the organs of chickens (A) and ducks (B) at day 3 p.i. inoculated with 10⁶ EID₅₀ of the test virus. The data shown are the mean with SD of the titers from three chickens or ducks. The dashed line indicates the lower limit of detection.

FIG 7

Transmission of H10N4 and H10N8 viruses in chickens and ducks. Groups of SPF chickens and ducks (n = 3) were inoculated intranasally with 10⁶ EID⁵⁰ virus in a volume of 200 μL. Three naive chickens or ducks of the contacted group were housed in the same isolator as the contact birds at 24 hpi. The oropharyngeal and cloacal swabs of the animals were suspended in 1 mL of PBS and titrated for viral shedding in eggs. (A to C) Transmission study of H10N4 and H10N8 viruses in chickens. (D to F) Transmission study of H10N4 and H10N8 viruses in ducks. The dashed line indicates the lower detection limit.