Towards a better understanding of the novel avian-origin H7N9 influenza A virus in China

Abstract

Recently, a highly dangerous bird flu has infected over 130 patients in China, and the outbreak was attributed to a novel avian-origin H7N9 virus. Here, we performed a systematic analysis of the virus. We clarified the controversial viewpoint on neuraminidase (NA) origin and confirmed it was reassorted from Korean wild birds with higher confidence, whereas common ancestors of pathogenic H7N9 genes existed only one or two years ago. Further analysis of NA sequences suggested that most variations are not drug resistant and current drugs are still effective for the therapy. We also identified a potentially optimal 9-mer epitope, which can be helpful for vaccine development. The interaction of hemagglutinin (HA) and human receptor analog was confirmed by structural modeling, while NA might influence cellular processes through a PDZ-binding motif. A simplified virus infection model was proposed. Taken together, our studies provide a better understanding of the newly reassorted H7N9 viruses.

Figure 1. Phylogenetic analyses of H7N9 virus genes.

The MP trees were present for (A) HA and (B) NA. (C) The NJ tree was visualized for PB1. The HPAI viruses in patients were marked in red, while HPAI viruses from non-human samples were marked in blue. The nearest genes from LPAI viruses were shown in green. More detailed phylogenetic trees were shown in Supplementary Fig. S1.

Figure 2. A variation map of HPAI H7N9 genes in patient samples.

We analyzed variations between patients and non-pathogenic samples and among 4 patients, respectively. The variation patterns were also counted. (a) The benchmark HA sequence was taken from A/duck/Zhejiang/11/2011(H7N3); (b) Using identical positions of alignment results for 5 Korean wild bird H7N9 NA sequences as the background; (c) From A/chicken/Hebei/1102/2010(H5N2); (d) From A/chicken/Zhejiang/607/2011 (H9N2); (e) The statistics of variation patterns confirmed that H7N9 virus in A/Shanghai/1/2013 evolves more rapidly.

Figure 3. The structure modeling of NA-drug interactions.

(a) The original NA-Oseltamivir complex from 2QWK; Other complex structures are modeled for H7N9 subtype, including (b) NA-Oseltamivir, (c) NA-Laninamivir and (d) NA-Zanamivir. The drug-interacting residues (≤ 4 Å) were highlighted and labeled.

Figure 4. The molecule docking of HLA-B*4405, DM1-TCR and predicted epitopes.

The key interacting residues in epitopes were labeled, while their binding sites in HLA-B*4405 or DM1-TCR were highlighted in red. (a) HLA-B*4405/epitope 88; (b) HLA-B*4405/epitope 353; (c) DM1-TCR/HLA-B*4405/epitope 88; (d) DM1-TCR/HLA-B*4405/epitope 353.

Figure 5. The analyses of PDZ-binding in influenza A virus.

(a) The predicted PDZ-binding motifs in HPAI H7N9 and H5N1; (b) Interacting residues in NS1-binding PDZ domain (red); (c) The PDZ-binding motif in H7N9 NA (red); (d) Secondary structures of H7N9 NA C-terminus.

Figure 6. The sequence alignment of HPAI H7N9 NA with the sequences of A/mallard/Czech Republic/13438-29K/2010 (JF789604, H11N9) and A/wild bird/Korea/A3/2011 (JN244223, H7N9).

Figure 7. A “three-step reassortant” model of avian-origin H7N9 viruses.

The six internal genes were reassorted from brambling and poultries in the Yangtze river delta before November 2012. HA of H7N3 was reassorted with the internal genes in poultries after November 2012. The final step for the reassortment of H7N9 NA from Korean wild bird occurred in Shanghai before 19/02/2013.

Figure 8. The modeled HA structures in complex with (a) avian receptor analog 3'SLN and (b) human receptor analog 6'SLN.

The sticks represent the analog (3'SLN or 6'SLN) while the lines represent the HA. The complexes of NA-analog for A/Netherlands/219/2003 and A/Anhui/2013 were aligned together for comparison. The backbone of HA was shown in grey, while binding residues were labeled and their C atoms were highlighted in green. The hydrogen bonds were shown in black for A/Netherlands/219/2003 and magenta for A/Anhui/2013.

Figure 9. A model of HPAI H7N9 virus infection.

The viruses enter human cells through the HA-6'SLN interaction, while both HA and NA further interact with PDZ domain proteins to influence biological processes.