N-Glycolylneuraminic Acid in Animal Models for Human Influenza A Virus

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: CMAH; N-acetylneuraminic acid; N-glycolylneuraminic acid; animal model; ferret; influenza; mouse; sialic acid linkage.

Figure 1

Structure of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc).

Figure 2

Visualization of the presence of α2,3 linked Neu5Ac (WT HA of A/Vietnam/1203/2004 (H5N1)), α2,3 linked Neu5Gc (Y161A mutant HA of A/Vietnam/1203/2004 (H5N1)), and α2,6 linked sialic acids (SNA) using AEC staining on lung tissue of mouse (C57BL/6), ferret, guinea pig, Syrian hamster, and domestic swine. Brown staining indicates binding of the HAs to the tissue and blue indicates the cells. Selected bronchioles (black arrows) and blood vessels (blue arrows) are indicated. Images are representative of two independent experiments.

Figure 3

H5 WT and HA-Y161A binding properties and infectivity in WT (C57BL/6) and CMAH^−/− mice. (A) A hemagglutination assay (n = 3) with canine and equine erythrocytes was performed using HAs of WT and Y161A mutants of—A/Vietnam/1203/2004 (H5VN) and A/Hong Kong/483/1997 (H5HK). (B) Tissue binding of the HA of H5HK WT and mutant Y161A on canine and equine trachea is visualized with AEC staining (representative for three independent assays), the scale bar represents 100 µm. (C) Mice lungs (WT and CMAH^−/− knockout) were stained in duplicate with anti-Neu5Gc IgY and binding was visualized with AEC staining. WT (C57BL/6) and CMAH^−/− knockout mice (n = 5) were infected with H5HK WT and HA-Y161A mutant virus and (D) the survival and (E) body weight curves are shown, together with the (F) PFU per gram at 3 dpi in the lungs (n = 5), *** indicates a p-value of 0.0007 in an unpaired two-tailed t-test. (G) Synthetic glycans printed on the microarray (n = 6), either without sialic acid (structures 1–3, light gray), with α2,6-linked Neu5Ac (4–6, black), α2,3-linked Neu5Ac (7–9, dark gray), α2,6-linked Neu5Gc (10–12, red) or α2,3-linked Neu5Gc (13–15, blue). Binding specifities of WT and Y161A mutant H5HK HAs were investigated.