Pathogenicity of Novel H3 Avian Influenza Viruses in Chickens and Development of a Promising Vaccine

Abstract

Since 2022, three cases of human infections of novel H3N8 avian influenza viruses (AIVs) have been confirmed in China. Given the potential for significant public health implications, the prompt detection and containment of the virus is particularly important. Comprehensive analyses were conducted of the complete viral gene sequences of five H3 subtype AIVs that were isolated from chickens, pigeons, and geese in live poultry markets in China in 2023. Four strains exhibited a high degree of homology with the H3N8 viruses responsible for human infections in 2022 and 2023. A subsequent study was conducted to investigate the pathogenicity differences among multiple subtypes of the H3 AIVs in chickens. The study revealed that all infected chickens exhibited clinical signs and viral shedding. Notably, two H3N8 viruses, which were highly homologous to human strains, demonstrated significant differences in adaptability to chickens. The goose-derived H3N5 strain displayed high adaptability to chickens and could replicate in multiple organs, with the highest titer in the cloaca. Additionally, a potential vaccine strain, designated CK/NT308/H3N3, was successfully developed that provided complete clinical protection and effectively prevented viral shedding against both H3N3 and H3N8 viruses. In conclusion, CK/NT308/H3N3 presents a promising vaccine candidate.

Keywords: H3 avian influenza viruses; chicken; protective effect; vaccine.

Figure 1

Phylogenetic trees illustrating the relationships among H3 AIV strains isolated in eastern China and the eight gene segments of human-derived strains. Phylogenetic trees of the H3, N3, and N8 genes are depicted in panels (A–C), respectively. The phylogenetic relationships among the PB2, PB1, and PA gene segments are illustrated in panels (D–F), respectively. Phylogenetic trees of the NP, M, and NS genes are provided in panels (G–I), respectively. The trees were constructed using the neighbor-joining method with MAGE 11 software with version 11.0.11. The strains isolated in this study are indicated by red solid circles, while AIVs identified as the cause of human infections in China are shown in blue italics. The strains marked in black were obtained from the Global Initiative on Sharing Avian Influenza Data and the Influenza Virus Resource of the National Center for Biotechnology Information. The scale represents the mean amino acid substitutions at each site.

Figure 2

Pathogenicity of H3 subtype AIVs in chickens. (A,B) Shedding and replication of H3 virus in chickens. SPF chickens were inoculated with strains CK/P1027/H3N8, CK/G1106/H3N8, PG/J1247/H3N3, and GS/E1281/H3N5. Pharyngeal and cloacal swabs were collected at 2-day intervals from dpi 3 to 11, and the virus titer in eggs was determined (A). Concurrently, organ samples were collected on dpi 3, and the virus titer was determined in eggs (B). The columns represent the mean virus titer, while the error bars indicate the standard deviation. OP and CL refer to oropharyngeal and cloacal swabs, respectively. The dotted line on the y-axis indicates the lower limit of virus detection. Histological examinations of selected representative organs were conducted by the staining of microscopic sections with hematoxylin and eosin. From left to right, these included the lungs, trachea, and kidneys following infection with CK/G1106/H3N8, as well as the spleen and bursa of Fabricius following infection with GS/E1281/H3N5 (C).

Figure 3

The level of immune protection provided by the vaccine. Antibody titers on dpi 21 with inactivated vaccines derived from the CK/NT308/H3N3 strain were assessed against the strains CK/P1027/H3N8 (A), CK/G1106/H3N8 (B), PG/J1247/H3N3 (C), and CK/NT308/H3N3 (D). On post-vaccination day 3, the viral titer (E) in eggs was quantified. Concurrently, oropharyngeal and cloacal swabs were obtained from the chickens on dpi 3, 5, 7, 9, and 11, and the viral titers (F) in eggs were determined. Microscopic sections of selected representative organs were stained with hematoxylin and eosin. From left to right, the examined organs included the lungs, trachea, kidneys, spleen, and bursa of Fabricius (G).