Novel Sequence-Based Mapping of Recently Emerging H5NX Influenza Viruses Reveals Pandemic Vaccine Candidates

<div><p>Recently, an avian influenza virus, H5NX subclade 2.3.4.4, emerged and spread to North America. This subclade has frequently reassorted, leading to multiple novel viruses capable of human infection. Four cases of human infections, three leading to death, have already occurred. Existing vaccine strains do not protect against these new viruses, raising a need to identify new vaccine candidate strains. We have developed a novel sequence-based mapping (SBM) tool capable of visualizing complex protein sequence data sets using a single intuitive map. We applied SBM on the complete set of avian H5 viruses in order to better understand hemagglutinin protein variance amongst H5 viruses and identify any patterns associated with this variation. The analysis successfully identified the original reassortments that lead to the emergence of this new subclade of H5 viruses, as well as their known unusual ability to re-assort among neuraminidase subtypes. In addition, our analysis revealed distinct clusters of 2.3.4.4 variants that would not be covered by existing strains in the H5 vaccine stockpile. The results suggest that our method may be useful for pandemic candidate vaccine virus selection.</p></div

Variation within the B cell epitopes of recent clade 2.3.4.4 H5NX viruses.

<p><b>(A)</b> SBM was performed on truncated sequences contain only amino acids within B cell epitopes. Sequences colored by subtype. Crossed-diamond symbols indicate human infection sequences. (B) Phylogenetic tree of 2.3.4.4 subclade HA. Labels were added for comparison with Fig 4A.</p

Amino acid substitutions in clade 2.3.4.4 H5NX viruses.

<p>(A) Number of unique amino acids found at each position (1–568) across H5NX viruses. HA1 and HA2 regions of the hemagglutinin protein are indicated in box. (B) Mapping of unique amino acids onto 3D reconstruction of H5N1 HA. Number of unique amino acids found at each amino acid position is colored as depicted in legend.</p

Comprehensive analysis of H5 virus Hemagglutinin.

<p>(A) Phylogenetic tree of influenza hemagglutinin proteins constructed from complete set of H5 HA sequences. Nodes were colored by continent of isolation. (B) SBM analysis on all publically available HA sequences for H5 viruses, sequences colored by continent of isolation. (C) Sequences colored by clade, only clades with >50 sequences were colored. (D) Sequences color by NA subtype.</p

Epitope Amino Acid Differences Contributing to PC1 and PC2 position for 2.3.4.4. H5NX Strains.

<p>Epitope Amino Acid Differences Contributing to PC1 and PC2 position for 2.3.4.4. H5NX Strains.</p

SBM Analysis of clade 2.3.4.4 H5NX viruses.

<p>SBM analysis restricted to virus HA sequences belonging to clade 2.3.4.4. (A) HA sequences colored by country of isolation. (B) Sequences colored by NA subtype. (C) Sequences colored by year of isolation as a continuum from green to red (2008–2015 respectively). (D) Sequences colored by species in which virus was isolated.</p

Additional file 2: of Comparative analysis of anti-viral transcriptomics reveals novel effects of influenza immune antagonism

Figures showing normalized levels of IFNB1 (Figure S1) and identifiability of model parameters using the proposed estimation protocol (Figure S2)

Results of the simulations of the time course of <i>T. retortaeformis</i> infection from the co-infection.

<p>Activity profiles (the probability of the node being in an ON state at a given time-step) are reported for: <b>A</b>- Third stage infective larvae (L3) and adults. <b>B</b>- Cytokines, IFNγ, IL4 and IL10 in the duodenum. <b>C</b>- Mucus antibodies against adult helminths. <b>D</b>- Peripheral eosinophils and neutrophils. Note that the IFNγ concentration range is between 0–2 to describe additional non-immune mediated activation of that node by the tissue damage (details in the <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002345#s2" target="_blank">Results</a>).</p

Parasite activity at the 20^th time step from simulations where network nodes were individually knocked out (from 100 replicates).

<p><b>A</b>- <i>B. bronchiseptica</i> in single infection. <b>B</b>- <i>B. bronchiseptica</i> in co-infection. <b>C</b>- <i>T. retortaeformis</i> in single infection. <b>D</b>- <i>T. retortaeformis</i> in co-infection. Explanation of the abbreviations is reported in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002345#pcbi-1002345-g001" target="_blank">Figure 1</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002345#pcbi-1002345-g002" target="_blank">Figure 2</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002345#pcbi.1002345.s003" target="_blank">Text S1</a>.</p

Summary of linear mixed effect model (LME) between <i>T. retortaeformis</i> abundance (worm/small intestine length) as a response, and infection type (single or co-infection), day post infection (DPI) and organ location (from the duodenum -SI1- to the ileum -SI4-), as independent variables.

<p>The random effect of the host identity code (ID) and the autocorrelation effect (AR-1) of sampling different organs of the same host are also reported.</p