N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses

Ruyi Gao,Min Gu,Daxin Peng,Kaituo Liu,Shunlin Hu,Xinan Jiao,Xiuli Li,Liwei Shi,Xiufan Liu,Xiaoquan Wang,Sujuan Chen,Jiao Hu,Xiaowen Liu

doi:10.1186/s13567-020-00879-6

Abstract

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Highlights

The Asian lineage of H5N1 (H5NX) subtype highly pathogenic avian influenza virus (HPAIV) was first isolated in 1996 from diseased geese in southern China [1]
Glycosylation at site 158 does not affect the antigenicity of the H5N6 virus Two H5N6 variants used in this study have distinct glycosylation sites at 158 of the HA proteins
Glycosylation at site 158 enhances pathogenicity of the H5N6 virus in mice We investigated if glycosylation at 158 contributed to the pathogenicity of the H5N6 viruses in mice

Summary

Introduction

The Asian lineage of H5N1 (H5NX) subtype highly pathogenic avian influenza virus (HPAIV) was first isolated in 1996 from diseased geese in southern China [1]. The genetic composition of H5N6 HPAIV is extremely complex. It is comprised of as many as 34 genotypes, Gao et al Vet Res (2021) 52:8 including a genotype whose internal gene cassette is entirely derived from the H9N2 avian influenza virus (AIV) [6, 7]. In addition to avian species including poultry, waterfowl, and wild birds, some mammals such as pigs and cats can be infected [8, 9]. Except for H5N1, the H5N6 subtype of HPAIV is currently the only H5 subtype that can infect humans [10]. As of June 2020, a total of 24 cases of human infection with H5N6 HPAIV have been reported, including eight deaths [4]. H5N6 HPAIV poses a realistic threat to public health

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Conclusion