Truncation or Deglycosylation of the Neuraminidase Stalk Enhances the Pathogenicity of the H5N1 Subtype Avian Influenza Virus in Mallard Ducks

Abstract

H5N1 subtype avian influenza virus (AIV) with a deletion of 20 amino acids at residues 49-68 in the stalk region of neuraminidase (NA) became a major epidemic virus. To determine the effect of truncation or deglycosylation of the NA stalk on virulence, we used site-directed mutagenesis to insert 20 amino acids in the short-stalk virus A/mallard/Huadong/S/2005 (SY) to recover the long-stalk virus (rSNA+). A series of short-stalk or deglycosylated-stalk viruses were also constructed basing on the longstalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylatedstalk viruses had smaller plaques, and increased thermal and low-pH stability, and a decreased neuraminidase activity when compared with the virus rSNA+. In a mallard ducks challenge study, most of the short-stalk or deglycosylated-stalk viruses showed increased pathological lesions and virus titers in the organ tissues and increased virus shedding in the oropharynx and cloaca when compared with the rSNA+ virus, while most of the short-stalk viruses, especially rSNA-20, showed higher pathogenicity than the deglycosylated-stalk virus. In addition, the short-stalk viruses showed a significantly upregulated expression of the immune-related factors in the lungs of the infected mallard ducks, including IFN-α, Mx1, and IL-8. The results suggested that NA stalk truncation or deglycosylation increases the pathogenicity of H5N1 subtype AIV in mallard ducks, which will provide a pre-warning for prevention and control of H5N1 subtype avian influenza in the waterfowl.

Highly pathogenic avian influenza virus of the A/H5N8 subtype, clade 2.3.4.4b, caused outbreaks in Kazakhstan in 2020

“…Three mutations in NA (N46K, N84S and T295M) deserve special mentioning because these mutations remove predicted N-linked glycosylation sites and thus are assumed to diminish glycosylation in the NA stem region. There are experimental studies showing that the abrogation of the NA glycosylation enhances the virulence, increases pathology in birds and mice ( Chen et al, 2020 ; Park et al, 2017 ).…”

Higher Viral Stability and Ethanol Resistance of Avian Influenza A(H5N1) Virus on Human Skin

“…Those results strongly suggest that the higher stability and EA resistance of the H5N1 subtype might depend on NA, a spike protein. Although several studies have focused on the relationship between the NA segment and virulence ( 42 , 43 ), to the best of our knowledge, no study has focused on the relationship between the NA segment and stability. Future studies focusing on the NA segment are expected to elucidate factors that determine the stability and help identify subtypes with high stability and a high risk for contact transmission.…”

The Pathobiology of H7N3 Low and High Pathogenicity Avian Influenza Viruses from the United States Outbreak in 2020 Differs between Turkeys and Chickens

“…Deletions in the NA stalk have been associated with poultry adaptation [ 14 , 15 , 16 , 17 , 64 ], outbreaks in the field [ 34 ], and experimental virus passage in gallinaceous birds [ 37 , 38 ]. Other studies showed that influenza viruses with different NA stalk lengths have different in vitro growth characteristics and plaque size [ 39 , 65 , 66 ] and differences in pathogenesis in ducks and chickens [ 39 , 63 ]. A study demonstrated that an amino acid deletion in the NA stalk can remove potential glycosylation sites, which may interfere in the protein’s function due to possible changes in the structure and consequently affect the immune response [ 67 ].…”