viruses Article N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus Cindy M. Spruit 1 , Nikoloz Nemanichvili 2 , Masatoshi Okamatsu 3 , Hiromu Takematsu 4 , Geert-Jan Boons 1,5 and Robert P. de Vries 1, *   Citation: Spruit, C.M.; Nemanichvili, N.; Okamatsu, M.; Takematsu, H.; Boons, G.-J.; de Vries, R.P. N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus. Viruses 2021, 13, 815. https://doi.org/10.3390/v13050815 Academic Editor: Jessica A. Belser Received: 25 March 2021 Accepted: 28 April 2021 Published: 1 May 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1 Department of Chemical Biology & Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3584 CG Utrecht, The Netherlands; c.m.spruit@uu.nl (C.M.S.); g.j.p.h.boons@uu.nl (G.-J.B.) 2 Division of Pathology, Department of Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands; n.nemanichvili@uu.nl 3 Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Hokkaido, Japan; influenza.pesti@gmail.com 4 Department of Molecular Cell Biology, Faculty of Medical Technology, Graduate School of Health Sciences, Fujita Health University, 1-98 Dengakugakubo, Kutsukake, Toyoake 470-1192, Aichi, Japan; htakema@fujita-hu.ac.jp 5 Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA * Correspondence: r.vries@uu.nl Abstract: The first step in influenza virus infection is the binding of hemagglutinin to sialic acid- containing glycans present on the cell surface. Over 50 different sialic acid modifications are known, of which N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the two main species. Animal models with α2,6 linked Neu5Ac in the upper respiratory tract, similar to humans, are preferred to enable and mimic infection with unadapted human influenza A viruses. Animal models that are currently most often used to study human influenza are mice and ferrets. Additionally, guinea pigs, cotton rats, Syrian hamsters, tree shrews, domestic swine, and non-human primates (macaques and marmosets) are discussed. The presence of NeuGc and the distribution of sialic acid linkages in the most commonly used models is summarized and experimentally determined. We also evaluated the role of Neu5Gc in infection using Neu5Gc binding viruses and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) −/− knockout mice, which lack Neu5Gc and concluded that Neu5Gc is unlikely to be a decoy receptor. This article provides a base for choosing an appropriate animal model. Although mice are one of the most favored models, they are hardly naturally susceptible to infection with human influenza viruses, possibly because they express mainly α2,3 linked sialic acids with both Neu5Ac and Neu5Gc modifications. We suggest using ferrets, which resemble humans closely in the sialic acid content, both in the linkages and the lack of Neu5Gc, lung organization, susceptibility, and disease pathogenesis. Keywords: influenza; animal model; N-glycolylneuraminic acid; N-acetylneuraminic acid; CMAH; sialic acid linkage; mouse; ferret 1. Introduction Infection of humans by influenza A viruses starts at the epithelium cells in the upper respiratory tract, where the hemagglutinin (HA) on the outside of a virus particle binds to glycans with a terminal sialic acid. The terminal sialic acids can be linked through an α2,3 or α2,6 bond to the penultimate galactose. In humans, mainly α2,6 linked sialic acids are present in the upper respiratory tract. The expression of α2,3 and α2,6 linked sialic acids varies between species and tissues [1]. Another variable in the influenza receptor is the type of sialic acid, of which N- acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the main species (Figure 1). The majority of influenza A viruses use a glycan with a terminal Viruses 2021, 13, 815. https://doi.org/10.3390/v13050815 https://www.mdpi.com/journal/viruses