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

Sujuan Chen,Tao Qin,Xiufan Liu,Keji Quan,Dandan Wang,Daxin Peng,Yinping Du

doi:10.3389/fmicb.2020.583588

Sujuan Chen, Tao Qin + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2020.583588

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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 long-stalk virus, and then the characteristics and pathogenicity of the resulting viruses were evaluated. The results showed that most of the short-stalk or deglycosylated-stalk 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.

Highlights

Avian influenza virus (AIV) has a wide range of hosts and can be isolated from poultry, waterfowl, and some mammals, in which it displays different morbidities and mortalities (Wheatley and Kent, 2015)
The Chicken embryo fibroblasts (CEF) and duck embryo fibroblasts (DEF) cells were infected in triplicate with the recombinant viruses at an multiplicity of infection (MOI) of 0.01 in M199, and the medium was discarded at 1 h.p.i
Western blot results showed that compared with rSNA+, the molecular weight of NA from the recombinant virus was reduced with the degrees of stalk truncation or deglycosylation

Summary

INTRODUCTION

Avian influenza virus (AIV) has a wide range of hosts and can be isolated from poultry, waterfowl, and some mammals, in which it displays different morbidities and mortalities (Wheatley and Kent, 2015). The percentage of viruses with a short NA stalk dramatically increased from 1996 to 2012 among all avian isolates, including those from wild birds (Matsuoka et al, 2009) This has been reported previously in the NAs of avian influenza H2N2, H5N1, H6N1, H7N1, H7N9, H7N3, and H9N2 subtype viruses (Matrosovich et al, 1999; Banks et al, 2001; Campitelli et al, 2004; Matsuoka et al, 2009; Sorrell et al, 2010; Gao et al, 2013; Sun et al, 2013). We constructed a series of NA stalk-truncated or deglycosylated recombinant viruses by reverse genetics and determined their biological characteristics, including replication kinetics, stability under different conditions, neuraminidase activity, and pathogenicity in mallard ducks

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RESULTS

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