A single amino acid in the HA of pH1N1 2009 influenza virus affects cell tropism in human airway epithelium, but not transmission in ferrets

Abstract

The first pandemic of the 21(st) century, pandemic H1N1 2009 (pH1N1 2009), emerged from a swine-origin source. Although human infections with swine-origin influenza have been reported previously, none went on to cause a pandemic or indeed any sustained human transmission. In previous pandemics, specific residues in the receptor binding site of the haemagglutinin (HA) protein of influenza have been associated with the ability of the virus to transmit between humans. In the present study we investigated the effect of residue 227 in HA on cell tropism and transmission of pH1N1 2009. In pH1N1 2009 and recent seasonal H1N1 viruses this residue is glutamic acid, whereas in swine influenza it is alanine. Using human airway epithelium, we show a differential cell tropism of pH1N1 2009 compared to pH1N1 2009 E227A and swine influenza suggesting this residue may alter the sialic acid conformer binding preference of the HA. Furthermore, both pH1N1 2009 E227A and swine influenza multi-cycle viral growth was found to be attenuated in comparison to pH1N1 2009 in human airway epithelium. However this altered tropism and viral growth in human airway epithelium did not abrogate respiratory droplet transmission of pH1N1 2009 E227A in ferrets. Thus, acquisition of E at residue 227 was not solely responsible for the ability of pH1N1 2009 to transmit between humans.

Figure 1. Distribution of amino acids at position 227 in swine H1 Has.

Presence of alanine, glutamic acid and other amino acids at position 227 in 173 full-length HA sequences of pre 2009 North-American swine H1 influenza.

Figure 2. E195 shows a tropism preference for non-ciliated cells compared to E195 E227A and Ohio01 on HAE cultures.

a/c/e) HAE culture sections were probed with HA-Fc proteins from human or swine H1N1 influenza virus strains. Ciliated cells were identified using anti-acetylated α-tubulin (red), and the HA-Fc proteins were visualized with anti-human Fc (green). Images are representative of multiple probed sections. Both non-ciliated cells (white arrow) and ciliated cells (yellow arrow) were present on human airway epithelium sections. b/d/f) HAE cultures were infected with MOI of 0.1 for 16 hrs. with isogenic viruses containing the HA genes of E195, E195 E227A or Ohio01. Ciliated cells were identified using anti-acetylated α-tubulin (red) and infected cells were detected with anti-NP antibody (green). Images are representative of multiple probed sections. Both non-ciliated cells (white arrow) and ciliated cells (yellow arrow) were present on human airway epithelium sections. g-h) Cell tropism was quantified by blind counting >200 cells per sample and expressed as the ratio of non-ciliated to ciliated cells bound or infected.

Figure 3. E195 E227A and Ohio01 are attenuated in HAE cultures.

Comparison of multi-cycle virus growth in MDCK cells or HAE cultures inoculated at MOI of 0.001 with isogenic viruses based on the E195 genetic backbone but with different HA proteins; either E195, E195 E227A or Ohio01 HA. Viral titres were determined by standard plaque assay on MDCK cells. Data shown represent the mean titre and standard deviation of 3 cultures. Statistical differences between wild type E195 and E195 E227A or Ohio01 were determined using one-way ANOVA. *  =  p<0.05, **  =  p<0.01.

Figure 4. Transmission of wild type E195 in ferrets in respiratory droplet sentinels.

Viral titres in nasal wash samples of ferret pairs, each pair contained one donor (solid lines) and one respiratory droplet (dotted lines). Donors were inoculated with 10³ PFU E195. Samples were taken daily and viral titres were determined by standard plaque assay on MDCK cells.

Figure 5. Transmission of E195 E227A in contact and respiratory droplet sentinels.

Viral titres in nasal wash samples of ferret sets, each pair contained one donor (solid lines) and one respiratory droplet (dotted lines). Donors were inoculated with 10⁴ PFU E195 E227A. Samples were taken daily and viral titres were determined by standard plaque assay on MDCK cells.