Diversity of genotypes and pathogenicity of H9N2 avian influenza virus derived from wild bird and domestic poultry

Abstract

Introduction: The H9N2 subtype is a predominant avian influenza virus (AIV) circulating in Chinese poultry, forming various genotypes (A-W) based on gene segment origins. This study aims to investigate the genotypic distribution and pathogenic characteristics of H9N2 isolates from wild birds and domestic poultry in Yunnan Province, China.

Methods: Eleven H9N2 strains were isolated from fecal samples of overwintering wild birds and proximate domestic poultry in Yunnan, including four from common cranes (Grus grus), two from bar-headed geese (Anser indicus), and five from domestic poultry (Gallus gallus). Phylogenetic analysis was conducted to determine the genotypes, and representative strains were inoculated into Yunnan mallard ducks to assess pathogenicity.

Results: Phylogenetic analysis revealed that five isolates from domestic birds and one from a bar-headed goose belong to genotype S, while the remaining five isolates from wild birds belong to genotype A. These bird-derived strains possess deletions in the stalk domain of NA protein and the N¹⁶⁶D mutation of HA protein, typical of poultry strains. Genotype S H9N2 demonstrated oropharyngeal shedding, while genotype A H9N2 exhibited cloacal shedding and high viral loads in the duodenum. Both strains caused significant pathological injuries, with genotype S inducing more severe damage to the thymus and spleen, while genotype A caused duodenal muscle layer rupture.

Discussion: These findings suggest that at least two genotypes of H9N2 are currently circulating in Yunnan, and Yunnan mallard ducks potentially act as intermediaries in interspecies transmission. These insights highlight the importance of analyzing the current epidemiological transmission characteristics of H9N2 among wild and domestic birds in China.

Keywords: H9N2 AIV; bar-headed goose; common crane; genotype A; genotype S; pathologic analysis; phylogenetic analysis.

Figure 1

Phylogenetic analysis of the HA gene from H9N2 AIV. The phylogenetic tree is generated with IQ-TREE software employing the maximum likelihood (ML) method. Node support is evaluated through 1,000 bootstrap replicates, and support values exceeding 70% are indicated at the corresponding nodes. The 11 H9N2 strains sequenced in this study are denoted by red triangles, while reference sequences for each clade are emphasized in bold. Branch-specific nomenclature is detailed to the right of the tree.

Figure 2

Genetic diversity of the 11 H9N2 AIV isolated in Yunnan Province, China. Four H9N2 strains isolated from common crane (CC-3, CC-6, CC-7 and CC-11) and one from bar-headed goose (BHG-21) are classified as genotype A, having acquired all eight gene segments from the BJ/94-like lineage. Conversely, five stains from chickens (CK-54, CK-59, CK-68, CK-74 and CK-75) and the one other strain from a bar-headed goose (BHG-8) are categorized as genotype S, representing a triple- reassortment derived from the BJ/94-like, G1-like, and F/98-like lineages. In the depiction, ellipses represent the AIV strains, with each of the eight horizontal segments corresponding to a distinct viral gene: polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix proteins (M), and non-structural proteins (NS), respectively. Each color signifies a specific viral lineage, with BJ/94-like in blue, G1-like in green, and F/98-like in red.

Figure 3

The changes in body weight of Yunnan mallard ducks challenged with H9N2 over a period of 14 dpi (days post inoculation). Body weights are graphed as the fold change, comparing them to the initial values on the day of inoculation (0 dpi). The genotype A and genotype S inoculation groups consisted of ducks intranasally inoculated with the CC-3 and CK-74 strains, respectively. The control group consisted of ducks inoculated with allantoic fluid without the virus via the same route.

Figure 4

Virus loads in oropharyngeal, cloacal swabs and various organs of Yunnan mallard ducks challenged with genotype A and genotype S H9N2 AIV. Oropharyngeal and cloacal swab samples are collected from genotype S (A) and genotype A (B) H9N2 AIV inoculation groups at 1 dpi (days post inoculation), 3 dpi, 4 dpi, 7 dpi, 10 dpi, and 14 dpi. Tissue samples are obtained from four ducks in each group at 4 dpi (C) and 14 dpi (D). The genotype A inoculation group and genotype S inoculation group are treated with CC-3 and CK-74 strains, respectively, while control group is not exposed to any virus. Virus loads are determined based on viral RNA levels expressed as equivalents of EID₅₀ (eqEID₅₀). The eqEID₅₀ values are obtained by correlating viral titers to Ct values through rRT-PCR for the M gene, using viral standards with known tenfold dilution viral titers ranging from 10^6.0 to 10^0.0 EID₅₀ per 0.2 mL in egg allantoic fluid. The standards show the limit of detection at 10^0.22 EID₅₀ for genotype S strain and 10^1.04 EID₅₀ for genotype A strain. Samples with virus loads below the limit of detection are assigned values of 0.22 log eqEID₅₀ for genotype S inoculation group and 1.04 log eqEID₅₀ for genotype A inoculation group, respectively. Two-way ANOVA is performed using GraphPad Prism software version 9.5.0. Significance levels are indicated by symbols: ^***for p < 0.001, ^**for p < 0.01, and ^*for p < 0.05.

Figure 5

Macroscopic and microscopic pathological lesions observed in the eight tissue organs from Yunnan mallard duck challenged with genotype A and genotype S H9N2 AIV at 4 days post inoculation. (A–C) Illustrate the gross lesions (Scale bar = 1 cm), while (a–c) illustrate the corresponding microscopic lesions (Scale bar = 10 μm). Tissues presented in (A, a) originate from ducks in the control group, which are inoculated with virus-free chick embryo amniotic fluid. Conversely, tissues shown in (B, b, C, c), are derived from ducks in the genotype S and genotype A inoculation groups, treated with CK-74 and CC-3 strains, respectively. Notable microscopic injuries are marked including hepatocellular steatosis (black triangle), hydropic degeneration (green triangle), inflammatory infiltration (green pentagram), hyperemia (black pentagram), tracheal ciliated epithelium desquamation (black diamond-shaped), and duodenal muscle layer rupture (green diamond-shaped).