Functional Balance between the Hemagglutinin and Neuraminidase of Influenza A(H1N1)pdm09 HA D222 Variants

Abstract

D222G/N substitutions in A(H1N1)pdm09 hemagglutinin may be associated with increased binding of viruses causing low respiratory tract infections and human pathogenesis. We assessed the impact of such substitutions on the balance between hemagglutinin binding and neuraminidase cleavage, viral growth and in vivo virulence.Seven viruses with differing polymorphisms at codon 222 (2 with D, 3 G, 1 N and 1 E) were isolated from patients and characterized with regards hemagglutinin binding affinity (Kd) to α-2,6 sialic acid (SAα-2,6) and SAα-2,3 and neuraminidase enzymatic properties (Km, Ki and Vmax). The hemagglutination assay was used to quantitatively assess the balance between hemagglutinin binding and neuraminidase cleavage. Viral growth properties were compared in vitro in MDCK-SIAT1 cells and in vivo in BALB/c mice. Compared with D222 variants, the binding affinity of G222 variants was greater for SAα-2,3 and lower for SAα-2,6, whereas that of both E222 and N222 variants was greater for both SAα-2,3 and SAα-2,6. Mean neuraminidase activity of D222 variants (16.0 nmol/h/106) was higher than that of G222 (1.7 nmol/h/106 viruses) and E/N222 variants (4.4 nmol/h/106 viruses). The hemagglutination assay demonstrated a deviation from functional balance by E222 and N222 variants that displayed strong hemagglutinin binding but weak neuraminidase activity. This deviation impaired viral growth in MDCK-SIAT1 cells but not infectivity in mice. All strains but one exhibited low infectious dose in mice (MID50) and replicated to high titers in the lung; this D222 strain exhibited a ten-fold higher MID50 and replicated to low titers. Hemagglutinin-neuraminidase balance status had a greater impact on viral replication than hemagglutinin affinity strength, at least in vitro, thus emphasizing the importance of an optimal balance for influenza virus fitness. The mouse model is effective in assessing binding to SAα-2,3 but cannot differentiate SAα-2,3- from SAα-2,6- preference, nor estimate the hemagglutinin-neuraminidase balance in A(H1N1)pdm09 strains.

Sialic acid receptors: focus on their role in influenza infection

“…The first marker of pathogenicity and severe clinical outcome has been associated to a single point mutation D225G at the RBS of the HA, which has been detected in viruses from several countries around the world. [30][31][32][33][34] Studies using experimental in vivo models analyzing the pathogenic effect of the mutation D225G are controversial; [34][35][36][37] foremost, the amino acid residue 225 has shown polymorphism for 225D/G/N in the HA of the H1N1pdm09 virus. 38,39 Overall, viruses with 225G phenotype showed changes in the virus receptor-binding profile and the cell/tissue tropism and dual binding for SAα2,6 and SAα2,3.…”

“…On the contrary, viruses with 225N showed preference for SAα2,6, though they showed reduced ability to infect human tracheobronchial epithelial cells, an effect that was compensated with a supplementary mutation S185N, expanding their cell tropism, increasing the number of infected cells, and likely, in consequence, the severity of the infection and clinical outcome of the patient. 34,36,40,41 The evolution of the H1N1pdm09 virus has been followed in detail worldwide; this has been the basis for the selection of vaccine viruses, as well to monitor changes at the antigenic sites, or changes in HA that result in the selection of viruses with better receptor-binding properties. Eight monophyletic groups have been identified based on the sequence variation of the HA1 domain of the pH1N1 virus, actually with group 6B.1 being the most represented with viruses from around the globe.…”

Changes in the Length of the Neuraminidase Stalk Region Impact H7N9 Virulence in Mice

“…Viruses containing short-stalk NA were shown to have altered rates of virus growth, virulence, and transmission in chickens and mammals (9,12,13,(15)(16)(17), in which the functional balance between HA and NA in the influenza virion was found to be a critical determinant (18)(19)(20)(21)(22)(23). Hence, NA stalk length, levels of glycosylation on HA, and mutations in the HA and NA antigens represent potential evolutionary strategies for the control of HA-NA functional balance to optimize virus fitness (12,18,(20)(21)(22). The NA segment from the novel H7N9 virus was found to possess a 5-amino-acid deletion, but it is not known whether this deletion confers any particular biological advantage with regard to human infections.…”

The Flu Epidemic in Russia in the 2013 - 2014 Season: Etiology, Antigenic Properties of Hemagglutinin and Neuraminidase Activity

“…Эти данные свидетельствуют о том, что вирусы гриппа подтипов А(H3N2) и В практически полностью утратили сродство к клеткам и тканям куриных эмбрионов, в отличие от вирусов гриппа А(H1N1)pdm09, что указывает на разную рецеп-торную специфичность этих возбудителей [14,15]. Данные исследования указывают на отсутствие значимых изменений антигенных свойств молеку-лы НА вируса гриппа А(H1N1)pdm09 с момента его появления в человеческой популяции в 2009 году, несмотря на постепенное накопление точечных му-таций в соответствующем гене.…”