Glycosylation on Hemagglutinin Affects the Virulence and Pathogenicity of Pandemic H1N1/2009 Influenza A Virus in Mice

Yan Zhang,Meilin Jin,Huanchun Chen,Jiyue Cao,Yongtao Li,Jiping Zhu,Hongbo Zhou,Konrad C Bradley,Julian W Tang

doi:10.1371/journal.pone.0061397

Yan Zhang, Meilin Jin + Show 7 more

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https://doi.org/10.1371/journal.pone.0061397

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Journal: PloS one	Publication Date: Apr 24, 2013
Citations: 70	License type: CC BY 4.0

Affiliation: Huazhong Agricultural University, Emory University

Abstract

The two glycosylation sites (Asn142 and Asn177) were observed in the HA of most human seasonal influenza A/H1N1 viruses, while none in pandemic H1N1/2009 influenza A (pH1N1) viruses. We investigated the effect of the two glycosylation sites on viral virulence and pathogenicity in mice using recombinant pH1N1. The H1N1/144 and H1N1/177 mutants which gained potential glycosylation sites Asn142 and Asn177 on HA respectively were generated from A/Mexico/4486/2009(H1N1) by site-directed mutagenesis and reverse genetics, the same as the H1N1/144+177 gained both glycosylation sites Asn142 and Asn177. The biological characteristics and antigenicity of the mutants were compared with wild-type pH1N1. The virulence and pathogenicity of recombinants were also detected in mice. Our results showed that HA antigenicity and viral affinity for receptor may change with introduction of the glycosylation sites. Compared with wild-type pH1N1, the mutant H1N1/177 displayed an equivalent virus titer in chicken embryos and mice, and increased virulence and pathogenicity in mice. The H1N1/144 displayed the highest virus titer in mice lung. However, the H1N1/144+177 displayed the most serious alveolar inflammation and pathogenicity in infected mice. The introduction of the glycosylation sites Asn144 and Asn177 resulted in the enhancement on virulence and pathogenicity of pH1N1 in mice, and was also associated with the change of HA antigenicity and the viral affinity for receptor.

Highlights

Influenza A viruses are responsible for both seasonal epidemics and occasional pandemics in human
Based on the sequence comparing, we found that two glycosylation sites at Asn142 and Asn177 on the HA in most pre-2009 human seasonal influenza A H1N1 viruses, but not in 2009 pandemic H1N1/2009 influenza A (pH1N1) viruses (T144D, N177K)
Human embryonic kidney (293T) cells and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Cluture Collection (ATCC) and cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin

Summary

Introduction

Influenza A viruses are responsible for both seasonal epidemics and occasional pandemics in human. Under the pressure of the host’s immune system, the pandemic viruses need to change its antigenic structure (called antigenic drift) so as to escape from the defenses Such pressure and drift could be why influenza immunity is not always neutralizing, as minor variations to the virus render it ‘‘unknown’’ to the hosts’ adaptive immune response [2,3]. Glycosylation of HA can affect the host specificity, infectivity and virulence of an influenza strain either directly, by changing the biological properties of HA [7] or other mechanisms such as shielding antigenic regions of the protein [8,9,10,11], impeding the activation of the protein precursor HA0 via its cleavage into the disulfide-linked subunits HA1 and HA2 [12,13,14], or attenuating receptor binding capability [15,16,17,18,19]. Glycosylation at the 158N site and the receptor binding preference of the VN04 (H5N1) ca vaccine virus affected virus antigenicity and caused poor replication in the host [22]

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