The G57 genotype of the BJ/94-like H9N2 lineage exhibits increased replication and virulence in chickens compared to the G1 Middle East Group B lineage

Abstract

Avian influenza H9N2 viruses cause significant economic losses to the poultry industry and pose a public health risk due to their potential to reassort with other avian influenza viruses, generating strains with zoonotic and pandemic potential. Two major H9N2 lineages dominate globally: the G1 lineage (genotype G1-B), prevalent in the Middle East, Africa and the Indian subcontinent, and the BJ/94 lineage (predominantly genotype G57), dominant in China, Vietnam, South Korea, Indonesia, and the Far East. We investigated replication, transmission, and pathogenicity of representatives of these two lineages, linking genotype to phenotype. The G57 strain A/Ck/Vietnam/H7F-14-BN4-315/2014 (Vietnam/315) was more lethal to chicken embryos than the G1-B strain A/chicken/Pakistan/UDL-01/2008 (Pakistan/UDL-01). Vietnam/315 exhibited higher replication in both directly infected and contact chickens, with increased virus shedding from the oropharynx and cloaca. In contrast, Pakistan/UDL-01 virus was primarily shed from the oropharynx, highlighting differences in replication, tissue tropism and transmission. Gene analysis showed the M gene of Vietnam/315 enhanced replication in primary chicken kidney cells, whereas the PB2, HA, NA, and M genes promoted increased replication in Madin-Darby Canine Kidney cells. Both viruses showed preferential binding to avian-like receptors over human-like receptors. However, Vietnam/315, however, exhibited higher neuraminidase activity and a more acid-stable HA (pH fusion 5.2) than Pakistan/UDL-01 virus. These findings suggest G57 genotype viruses possess greater replication and transmission fitness than G1-B viruses in vivo, ex vivo and in vitro. Reassortment events involving G1-B strains acquiring G57 genes may enhance replication and virulence, potentially increasing the risk of animal and human infection. ImportanceH9N2 avian influenza viruses are widespread in poultry, resulting in significant economic losses and occasional human infections. Different genetic variants dominate in various regions, but their ability to cause disease and spread in poultry remains unclear. These viruses can exchange genes with other avian influenza strains, altering their infectivity and transmission. This study compared two major H9N2 genotypes: G1-B (common in the Indian subcontinent and the Middle East) and G57 (dominant in China and Vietnam). The G57 virus showed higher replication in laboratory tests and infected chickens, shedding more virus through the orofecal route. It also exhibited a stronger attachment to bird cells and had a more acid-stable HA, suggesting an increased potential for infection and spread. Our findings indicate that G57 genotype viruses are more infectious and adaptable in poultry than G1 viruses. Gene exchange with other avian influenza strains may generate more virulent viruses with increased transmission potential. This study supports the risk assessment of emerging strains and enhances disease mitigation strategies.