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

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

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

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

The PDZ-Binding Motif of Severe Acute Respiratory Syndrome Coronavirus Envelope Protein Is a Determinant of Viral Pathogenesis

<div><p>A recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) lacking the envelope (E) protein is attenuated <i>in vivo</i>. Here we report that E protein PDZ-binding motif (PBM), a domain involved in protein-protein interactions, is a major determinant of virulence. Elimination of SARS-CoV E protein PBM by using reverse genetics caused a reduction in the deleterious exacerbation of the immune response triggered during infection with the parental virus and virus attenuation. Cellular protein syntenin was identified to bind the E protein PBM during SARS-CoV infection by using three complementary strategies, yeast two-hybrid, reciprocal coimmunoprecipitation and confocal microscopy assays. Syntenin redistributed from the nucleus to the cell cytoplasm during infection with viruses containing the E protein PBM, activating p38 MAPK and leading to the overexpression of inflammatory cytokines. Silencing of syntenin using siRNAs led to a decrease in p38 MAPK activation in SARS-CoV infected cells, further reinforcing their functional relationship. Active p38 MAPK was reduced in lungs of mice infected with SARS-CoVs lacking E protein PBM as compared with the parental virus, leading to a decreased expression of inflammatory cytokines and to virus attenuation. Interestingly, administration of a p38 MAPK inhibitor led to an increase in mice survival after infection with SARS-CoV, confirming the relevance of this pathway in SARS-CoV virulence. Therefore, the E protein PBM is a virulence domain that activates immunopathology most likely by using syntenin as a mediator of p38 MAPK induced inflammation.</p></div

Effect of SARS-CoV E protein PBM on host gene expression.

<p>(A) Comparison of gene expression in lungs of infected mice using microarrays: wt versus mock-infected, mutPBM versus mock-infected and mutPBM versus wt-infected mice. Red spots indicate upregulated gene transcripts (fold change, >2) and green spots indicate downregulated gene transcripts (fold change, <−2). Only genes with a FDR of <0.01 were considered as candidate genes. (B) Candidate genes that were downregulated in mutPBM infected mice compared wt infected ones, were grouped on Gene Ontology terms. Numbers on the <i>x</i> axis indicate DAVID FDR values. (C) Selection of differentially expressed genes found in at least one functional group using DAVID software. The numbers indicate the fold change for each gene in mutPBM versus wt-infected mice. (D) Expression of inflammatory cytokines evaluated by RT-qPCR. Three independent experiments were analyzed with similar results in all cases. Error bars represent standard deviations of the mean of results from three experiments.</p

Virulence and viral growth of SARS-CoV-E-PBM-infected mice.

<p>16-week-old BALB/c mice were intranasally inoculated with 100,000 pfu of wt, ΔE, ΔPBM, mutPBM and potPBM viruses. Weight loss (A) and survival (B) were monitored for 10 days. Data represent two independent experiments with 5 mice per group. Differences in weight loss between attenuated and virulent viruses were statistically significant (<i>P</i><0.01). (C) Viral titer in lungs was determined at 2 and 4 days post infection (n = 3, each day). Error bars represent standard deviations. Statistically significant data are indicated with one (<i>P</i><0.05) asterisk.</p

Lung pathology of mice infected with recombinant SARS-CoV-E-PBM mutants.

<p>16-week-old BALB/c mice were inoculated intranasally with 100,000 pfu of wt, ΔE, ΔPBM, mutPBM and potPBM viruses. (A) Gross pathology of mouse lungs infected with recombinant viruses at 2 and 4 dpi. (B) Weight of left lungs excised from infected mice sacrificed at the indicated days (n = 3, each day). Error bars represent standard deviations. (C) Lung tissue sections from mice infected with recombinant viruses were prepared at 2 and 4 dpi and stained with hematoxylin and eosin. Three independent mice per group were analyzed. (D and E) Pathology scoring in lung of mice infected with SARS-CoV recombinant mutants. Lungs were harvested at 2 and 4 days and scored in a blinded fashion using 3 mice per condition on a scale of 0 (none) to 3 (severe), estimated according to previously described procedures <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004320#ppat.1004320-WohlfordLenane1" target="_blank">[34]</a>. Data are presented for edema (D) and cellular infiltrates (E). Mean values are reported and statistically significant data are indicated with two (<i>P</i><0.01) asterisks. Original magnification was 20x. Representative images are shown.</p