Functional analysis of DEPs from subset B.

<p>Pathway enrichment analysis for normalized log₂–transformed expression values of proteins that were differentially expressed in IAV-positive versus IAV-negative patients from subset B is presented. Pathway terms that were enriched for the DEPs from subset B are indicated on the y-axis, the number of DEPs in the respective pathway category is indicated on the x-axis. The p-value for the probability that the observed distribution of expression occurred by chance is represented by colors of bars. The cut-off value for the pathway p-values was chosen at 0.05.</p

Scatter plot of RPS6KA5 expression levels and viral load.

<p>Scatter plot of normalized log₂–transformed relative protein concentrations of RPS6KA5 which was the DEP that most strongly correlated with viral titers and log₂-transformed virus titers are presented for patients from subset B. Red line: linear regression model.</p

PCA analysis of normalized protein expression values of subset A.

<p>Principle component analysis (PCA) was performed with normalized log₂–transformed protein expression values from nasal washes for 18 samples from the subset A that was selected based viral load being higher than > 2⁸. The first two principal components are shown representing 42% and 16%, respectively, of the total variation. Healthy controls are labelled gray and IAV-positive samples are labelled red. In addition, sample identities (e.g., ID_4002) are shown. Horizontal and vertical axis represent principle component 1 and 2, respectively.</p

PCA analysis of normalized protein expression values.

<p>Principle component analysis (PCA) was performed with quantile normalized log₂–transformed protein expression values from nasal washes for all 24 samples. The first two principal components are shown representing 24% and 17%, respectively, of the total variation. Healthy controls are labeled gray and IAV-positive samples (in which influenza A or B was detected by PCR) are labeled red. In addition, sample identities (e.g. ID_4043) are shown. Horizontal and vertical axis represent principle component 1 and 2, respectively.</p

Respiratory Syncytial Virus Human Experimental Infection Model: Provenance, Production, and Sequence of Low-Passaged Memphis-37 Challenge Virus

<div><p>Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children and is responsible for as many as 199,000 childhood deaths annually worldwide. To support the development of viral therapeutics and vaccines for RSV, a human adult experimental infection model has been established. In this report, we describe the provenance and sequence of RSV Memphis-37, the low-passage clinical isolate used for the model's reproducible, safe, experimental infections of healthy, adult volunteers. The predicted amino acid sequences for major proteins of Memphis-37 are compared to nine other RSV A and B amino acid sequences to examine sites of vaccine, therapeutic, and pathophysiologic interest. Human T- cell epitope sequences previously defined by <i>in vitro</i> studies were observed to be closely matched between Memphis-37 and the laboratory strain RSV A2. Memphis-37 sequences provide baseline data with which to assess: (i) virus heterogeneity that may be evident following virus infection/transmission, (ii) the efficacy of candidate RSV vaccines and therapeutics in the experimental infection model, and (iii) the potential emergence of escape mutants as a consequence of experimental drug treatments. Memphis-37 is a valuable tool for pre-clinical research, and to expedite the clinical development of vaccines, therapeutic immunomodulatory agents, and other antiviral drug strategies for the protection of vulnerable populations against RSV disease.</p></div

Predicted amino acid sequence for RSV Memphis-37 F protein and alignments.

<p>Alignments are as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone-0113100-g001" target="_blank">Figure 1</a>, but for the F protein. Features of interest are shaded or boxed as indicated below the sequence. The blue line marks an antigenic site A, which is targeted by the monoclonal antibody Palivizumab.</p

Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.

<p>Legend: *Differences between Memphis-37 and RSV A2 are bolded.</p><p>Comparison of human CD4 and CD8 T-lymphocyte epitope sequences between respiratory syncytial virus Memphis-37 and A2 strains.</p

Predicted amino acid sequence for RSV Memphis-37 G protein and alignments.

<p>Predicted amino acid sequence is shown for the RSV Memphis-37 G protein. Sequence was aligned with five subtype A viruses (including two laboratory strains) and four subtype B viruses (including two laboratory strains). Virus nomenclature and GenBank Accession numbers used in the alignment are: RSVA Nashville (JX069801.1), RSVA Denver (GU591769.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Kumaria1" target="_blank">[42]</a>), RSVA Milwaukee (JF920069.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-RebuffoScheer1" target="_blank">[43]</a>), RSVA VR-26 Long (AY911262.1, Laboratory strain), RSVA A2 (M74568.1, Laboratory strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Stec1" target="_blank">[15]</a>), RSVB Dallas (JQ582843.1), RSVB Milwaukee (JN032117.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-RebuffoScheer1" target="_blank">[43]</a>), RSVB 9320 (AY353550.1, B9320 Laboratory strain), RSV strain B1 (AF013254.1, B1 Laboratory strain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113100#pone.0113100-Karron1" target="_blank">[44]</a>). N-terminal variable region: a.a. 67–147, central conserved region: a.a. 148–198, C-terminal hypervariable region: a.a. 199–298, CX3C motif: a.a. 182–186. <u>RSV Memphis-37 sequencing methods</u>: For sequencing purposes, Memphis-37 was taken after five passages and was amplified once more in a T25 flask of HEp-2 cells. Briefly, virus was added to cells in DMEM/0.1% BSA for 1.5 hours. Medium was removed and replaced with EMEM/5% FCS for four days. A lysate was prepared and viral RNA was extracted using a Qiagen viral RNA mini kit. PCR reactions were performed using the TaKaRa One Step RNA PCR Kit (AMV) using 5 µl (145 ng/µl) of viral RNA extracted with Qiagen QIAmp Viral RNA Mini Kit. Forward and reverse oligonucleotides were prepared at concentrations of 100 µM and 1 µl of each pair was used in each reaction. Incubations were at 50°C for 30 min. and 94°C for 2 min. Then 40 cycles were run at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1.5 min. For each sequencing reaction, 40 ng PCR product were mixed with 3.2 pmoles oligonucleotide primer in a final volume of 12 µl and submitted to the Hartwell Center at St. Jude for Sanger sequencing. Sequences were edited and a contig was created in Vector NTI SeqMan. The consensus contig for Memphis-37 was imported into CLC Workbench and predicted amino acid sequences were aligned with other RSV sequences.</p

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33

<div><p>Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.</p></div

RSV induces robust, rapid IL-33 and IL-13 production in the lungs of neonates.

<p>(<b>a</b>) Cytokine protein levels of IL-33 and IL-13 in whole lung homogenates of RSV-infected neonate (5-day-old; NR) and adult (6-8-week-old; AR) mice (<i>n</i> = 4–10 per group) at different days (0–10) post-infection (dpi). (<b>b</b>) Cytokine protein levels of IL-33 detected in BAL at 1 dpi from NR and AR mice (<i>n</i> = 8–10 per group) compared to controls. (<b>c</b>) Gating strategy for determination of IL-33 expression by median fluorescence intensity (MFI) in epithelial cells (CD45^- EpCam⁺) with representative IL-33 MFI histogram (quantified in inset vs. fluorescence-minus-one (FMO) control (dotted line)). (<b>d</b>) Representative micrographs of <i>in situ</i> hybridization for IL-33 mRNA with red arrows to indicate positive staining cells (top), magnified inset (bottom), and quantification of IL-33 mRNA positive cells per unit area of lung. Scale bar = 100 μm. (<b>e</b>) Cytokine protein levels of IL-33 in whole lung homogenates of neonates infected with RSV or UV-RSV compared to control at 1 dpi. *<i>P</i> < 0.05 (Student’s <i>t</i>-test; <b>a</b>, <b>c</b>, <b>d</b>) (Two-way ANOVA; <b>b</b>) (One-way ANOVA; <b>e</b>). Data are representative of two (<b>a</b>, <b>c</b>) or 2 pooled (<b>b</b>, <b>d</b>, <b>e</b>) independent experiments (means ± s.e.m).</p