Acquisition of Innate Inhibitor Resistance and Mammalian Pathogenicity During Egg Adaptation by the H9N2 Avian Influenza Virus.

Chung-Young Lee,Jae-Hong Kim,Se-Hee An,Hyuk-Joon Kwon,Youn-Jeong Lee,Jun-Gu Choi

doi:10.3389/fmicb.2018.01939

Chung-Young Lee, Jae-Hong Kim + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2018.01939

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Abstract

An H9N2 avian influenza A virus (AIV), A/chicken/Korea/01310/2001 (01310-CE20), was established after 20 passages of influenza A/chicken/Korea/01310/2001 (01310-CE2) virus through embryonated chicken eggs (ECEs). As a result of this process, the virus developed highly replicative and pathogenic traits within the ECEs through adaptive mutations in hemagglutinin (HA: T133N, V216G, and E439D) and neuraminidase (NA: 18-amino acid deletion and E54D). Here, we also established that 01310-CE20 acquired resistance to innate inhibitors present in the egg white during these passages. To investigate the role of egg-adapted mutations in resistance to innate inhibitors, we generated four PR8-derived recombinant viruses using various gene combinations of HA and NA from 01310-CE2 and 01310-CE20 (rH2N2, rH2N20, rH20N2, and rH20N20). As expected, rH20N20 showed significantly higher replication efficiency in MDCK cells and mouse lungs, and demonstrated greater pathogenicity in mice. In addition, rH20N20 showed higher resistance to innate inhibitors than the other viruses. By using a loss-of-function mutant and receptor-binding assay, we demonstrated that a T133N site directed mutation created an additional N-glycosite at position 133 in rH20N20. Further, this mutation played a crucial role in viral replication and resistance to innate inhibitors by modulating the binding affinities to avian-like and mammalian-like receptors on the host cells and inhibitors. Thus, egg-adapted HA and NA may exacerbate the mammalian pathogenicity of AIVs by defying host innate inhibitors as well as by increasing replication efficiency in mammalian cells.

Highlights

Influenza A virus (IAV) has two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on its enveloped surface, and these proteins play a crucial role in invasion and budding, respectively, in host cells
It has been previously reported that innate inhibitors prevent hemagglutination of IAVs in normal allantoic fluid (Svedmyr, 1949), and we hypothesized that these inhibitors might influence the selection pressure during egg adaptation of AIVs
There are innate inhibitors hindering the hemagglutination of AIVs in normal chicken allantoic fluid

Summary

Introduction

Influenza A virus (IAV) has two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on its enveloped surface, and these proteins play a crucial role in invasion and budding, respectively, in host cells. During adaptation to terrestrial birds, the H5N1 avian influenza A virus (AIV) with additional N-glycans near the HA RBS balanced its relatively low HA binding affinity by decreasing its NA activity via NA stalk deletion (Matsuoka et al, 2009). These mutations facilitated the balance of HA-NA activities by increasing the replication efficiency and pathogenicity of AIVs in mammalian hosts (Matsuoka et al, 2009; Zhao et al, 2017). It has been postulated that IAVs containing highly glycosylated HAs may be less pathogenic and have reduced replicative efficacy due to their increased susceptibility to C-type lectins (Matrosovich et al, 1998; Vigerust et al, 2007)

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