Hemagglutinin 222D/G Polymorphism Facilitates Fast Intra-Host Evolution of Pandemic (H1N1) 2009 Influenza A Viruses

Abstract

The amino acid substitution of aspartic acid to glycine in hemagglutinin (HA) in position 222 (HA-D222G) as well as HA-222D/G polymorphism of pandemic (H1N1) 2009 influenza viruses (A(H1N1)pdm09) were frequently reported in severe influenza in humans and mice. Their impact on viral pathogenicity and the course of influenza has been discussed controversially and the underlying mechanism remained unclarified. In the present study, BALB/c mice, infected with the once mouse lung- and cell-passaged A(H1N1)pdm09 isolate A/Jena/5258/09 (mpJena/5258), developed severe pneumonia. From day 2 to 3 or 4 post infection (p.i.) symptoms (body weight loss and clinical score) continuously worsened. After a short disease stagnation or even recovery phase in most mice, severity of disease further increased on days 6 and 7 p.i. Thereafter, surviving mice recovered. A 45 times higher virus titer maximum in the lung than in the trachea on day 2 p.i. and significantly higher tracheal virus titers compared to lung on day 6 p.i. indicated changes in the organ tropism during infection. Sequence analysis revealed an HA-222D/G polymorphism. HA-D222 and HA-G222 variants co-circulated in lung and trachea. Whereas, HA-D222 variant predominated in the lung, HA-G222 became the major variant in the trachea after day 4 p.i. This was accompanied by lower neutralizing antibody titers and broader receptor recognition including terminal sialic acid α-2,3-linked galactose, which is abundant on mouse trachea epithelial cells. Plaque-purified HA-G222-mpJena/5258 virus induced severe influenza with maximum symptom on day 6 p.i. These results demonstrated for the first time that HA-222D/G quasispecies of A(H1N1)pdm09 caused severe biphasic influenza because of fast viral intra-host evolution, which enabled partial antibody escape and minor changes in receptor binding.

Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

“…To investigate the influence of rNanA and rNanB on virus receptors, we used the lectins MAA and SNA, respectively, to detect SAα2-3Gal and SAα2-6Gal on the cell surface. Both receptors of influenza A virus could be identified on A549 ( Figures 2A,B ) and MDCK cells (Supplementary Figure 5 ), which is consistent with previously published data ( Kumari et al, 2007 ; Bauer et al, 2012 ; Seidel et al, 2014 ). SA α2,3-Gal and SA α2,6-Gal were strikingly reduced by rNanA at 1:100 and 1:1,000 dilutions when compared to the untreated cell control ( Figures 2A,B ).…”

“…High amounts of rNanA and rNanB removed the SA from A549 and MDCK cell surfaces that are used as viral receptors thereby compromising Jena/8178 replication. Influenza virus A(H1N1)pdm09 isolates preferentially attach to SA α2,6-Gal ( Stevens et al, 2006 ; Childs et al, 2009 ; Seidel et al, 2014 ). Therefore, removal of SA α2,6-Gal and/or SA α2,3-Gal by pneumococcal NAs ( Xu et al, 2011 ) impairs or even blocks the replication of Jena/8178.…”

“…Confluent A549 or MDCK cells were incubated for 48 h at 37°C with 10-fold diluted NanA or NanB. After methanol fixation the effect of pneumococcal NAs on cell surface SA was investigated by immunocytochemical staining with a DIG glycan differentiation kit (F. Hoffmann-La Roche AG) as reported ( Seidel et al, 2014 ). DIG-labeled Sambucus nigra agglutinin (SNA) or Maackia amurensis agglutinin (MAA) recognizes SA α2,6-Gal and SA α2,3-Gal, respectively.…”

Differential Biphasic Transcriptional Host Response Associated with Coevolution of Hemagglutinin Quasispecies of Influenza A Virus

“…HA-D222G was also detected after adaptation of A(H1N1)pdm09 isolates in mice (Ilyushina et al, 2010 ; Seyer et al, 2012 ; Song et al, 2013 ). Experimental evidence for the association of coevolution of HA-222D/G quasispecies of the A(H1N1)pdm09 isolate Jena/5258/09 (Jena/5258) and severe influenza with biphasic pathology was provided by us recently (Seidel et al, 2014 ). The increasing amounts of the HA-G222 variant in the lung (~8% at day 1 p.i.…”

Staphylococcus aureus Lung Infection Results in Down-Regulation of Surfactant Protein-A Mainly Caused by Pro-Inflammatory Macrophages

“…HA-D222-mpJena/5258 was obtained after three plaque-purification steps [27] from the lung of BALB/c mice infected with the A/H1N1/pdm09 influenza virus isolate A/Jena/5258/09 (kindly provided by Andi Krumbholz [28].…”