Ferrets exclusively synthesize Neu5Ac and express naturally humanized influenza A virus receptors

Abstract

Mammals express the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) on cell surfaces, where they act as receptors for pathogens, including influenza A virus (IAV). Neu5Gc is synthesized from Neu5Ac by the enzyme cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH). In humans, this enzyme is inactive and only Neu5Ac is produced. Ferrets are susceptible to human-adapted IAV strains and have been the dominant animal model for IAV studies. Here we show that ferrets, like humans, do not synthesize Neu5Gc. Genomic analysis reveals an ancient, nine-exon deletion in the ferret CMAH gene that is shared by the Pinnipedia and Musteloidia members of the Carnivora. Interactions between two human strains of IAV with the sialyllactose receptor (sialic acid--α2,6Gal) confirm that the type of terminal sialic acid contributes significantly to IAV receptor specificity. Our results indicate that exclusive expression of Neu5Ac contributes to the susceptibility of ferrets to human-adapted IAV strains.

Figure 1. Analysis of sialic acid in ferret and other mammalian species.

(a) Western blot analysis showing the absence/presence of Neu5Gc in serum samples (10 μg) when tested with anti-Neu5Gc antibody. (b) Western blot analysis of serum samples when tested with sialic-acid-specific lectin, SNA (c) Sugar analysis showing amount of Neu5Ac and Neu5Gc in serum samples. (d) Double immunostaining of Neu5Gc in mouse and ferret kidney tissue using chicken anti-Neu5Gc (Neu5Gc panels) and SubAB overlay followed by rabbit anti-SubA (SubAB panels). In a and b, F=ferret, H=human, B=bovine and M=mouse serum, respectively; + or − represent samples with or without neuraminidase treatment (see Supplementary Fig. 1 for full western blot images).

Figure 2. Genomic analysis of a deleted region in the ferretCMAH gene responsible for the absence of Neu5Gc in both ferrets and humans.

(a) Synteny in the CMAH region of mouse, human and cat genomes. (b) Representation of the CMAH region of the ferret genome characterized in this study. Black bars represent region spanned by BAC clones 446P7 and 182P23. Blue bars indicate location of probes used in screening BAC library. Arrows indicate position of primers used to amplify the PCR product spanning the CMAH-deletion region. (c) Comparison of the CMAH region in cat and ferret genomes. Seven-kb PCR product containing the CMAH-deleted region was amplified using primers CMAH_FOR and CMAH_REV, respectively. (*) indicates the start of the CMAH gene, (**) indicates individual exon(s) on the CMAH gene and (***) indicates the end of the CMAH gene. Images below show CMAH exon PCR products from exon 3, exon 5, exon 8, exon 11 and exon 12, from cat (C), dog (D), human (H) and ferret (F) genomic DNA.

Figure 3. Molecular analysis demonstrating the importance of sialic-acid (SA) type on HA and NA specificity.

(a) Chemical structures of SAα2,3Gal and SAα2,6Gal in which SA is either Neu5Ac (3′SL^Ac, 6′SL^Ac) or Neu5Gc (3′SL^Gc, 6′SL^Gc). (b) Haemagglutinin receptor specificity of human influenza A viruses pH1N1 and H3N2. ¹H NMR (bottom row) and STD NMR (above) spectra were obtained of an equimolar mixture of 2 mM 6′SL^Ac (Neu5Acα2,6Galβ1,4Glc) and 6′SL^Gc (Neu5Gcα2,6Galβ1,4Glc) with pH1N1 virus (A/California/04/2009, left panel) and H3N2 virus (A/Perth/16/2009, right panel), respectively. All NMR samples also contained a low concentration of oseltamivir carboxylate (50 μM, OC), a very potent nanomolar inhibitor of the viral neuraminidase to inhibit sialic-acid cleavage (Supplementary Fig. 4). Shown are only the axial and equatorial H3 protons (H3ax, H3eq) and the N-acetamido methyl (NHAc) and methylene (NHGc) protons of the sialic-acid moiety that are clearly distinguishable between 6′SL^Ac and 6′SL^Gc. The entire spectra are shown in Supplementary Fig. 5. (c) Neuraminidase substrate specificity of human influenza A viruses pH1N1 and H3N2. ¹H NMR spectroscopy was employed to follow the cleavage of sialosides (6′SL^Ac; 6′SL^Gc; 3′SL^Ac; 3′SL^Gc.) upon addition of pH1N1 virus (A/California/04/2009, left panel) and H3N2 virus (A/Perth/16/2009, right panel), respectively. The conversion rate was calculated using the absolute peak intensity of the sialyllactose H3eq-signals (±7.5% error) based on substrate depletion by a successive series of ¹H NMR spectra over 20 min at 37 °C (Supplementary Fig. 9).