The interdependencies of viral load, the innate immune response, and clinical outcome in children presenting to the emergency department with respiratory syncytial virus-associated bronchiolitis

<div><p>Respiratory syncytial virus (RSV) causes significant infant morbidity and mortality. For decades severe RSV-induced disease was thought to result from an uncontrolled host response to viral replication, but recent work suggests that a strong innate immune response early in infection is protective. To shed light on host-virus interactions and the viral determinants of disease, copy numbers of five RSV genes (NS1, NS2, N, G, F) were measured by quantitative real-time polymerase chain reaction (qPCR) in nasal wash samples from children with RSV-associated bronchiolitis. Correlations were sought with host cytokines/chemokines and biomarkers. Associations with disposition from the emergency department (hospitalized or sent home) and pulse oximetry O₂ saturation levels were also sought. Additionally, RNase P copy number was measured and used to normalize nasal wash data. RSV gene copy numbers were found to significantly correlate with both cytokine/chemokine and biomarker levels; and RNase P-normalized viral gene copy numbers (NS1, NS2, N and G) were significantly higher in infants with less severe disease. Moreover, three of the normalized viral gene copy numbers (NS1, NS2, and N) correlated significantly with arterial O₂ saturation levels. The data support a model where a higher viral load early in infection can promote a robust innate immune response that protects against progression into hypoxic RSV-induced lower respiratory tract illness.</p></div

RSV gene Copy Numbers (CN) are correlated highly and linearly, and correlations improve when data are normalized by RNase P CN.

<p>(A) N CN vs. NS2 CN showing the most correlated pair of CNs measured (r = 0.941; p<0.0001); F CN vs. NS2 CN showing the least correlated pair of CNs measured (r = 0.789; p<0.0001). (B) Normalized N CN vs. normalized NS2 CN (where normalized CN = RSV gene CN/ RNase P CN) showing the most correlated pair of normalized CNs measured (r = 0.963; p<0.0001); normalized F CN vs. normalized NS2 CN showing the least correlated pair of normalized CNs measured (r = 0.808; p<0.0001).</p

Spearman correlations between RNase P-normalized RSV gene copy numbers and biomarker activity levels.

<p>Spearman correlations between RNase P-normalized RSV gene copy numbers and biomarker activity levels.</p

Piedra-RSVgeneCNcorr_data-public registry.xls

Clinical, physiological, and molecular biological data from infants admitted to emergency department (ED) with physician-diagnosed bronchiolitis and positive for RSV.<br><br>Nasal wash samples collected (one from each infant) and analyzed for 1) RSV gene copy numbers (genes NS1, NS2, N, G, F) and host RNase P copy number by qPCR, 2) cytokine levels by multiplex human cytokine assay,  and 3) biomarker levels (caspase, LDH, MMP-7, MPO) by specific assay kits.<br><br>Infants were enrolled in study after obtaining written informed consent from a parent or legal guardian, and met all the inclusion criteria and none of the exclusion criteria. The inclusion criteria were the child was previously healthy and had a physician diagnosis of bronchiolitis in the ED. The exclusion criteria were co-morbid medical conditions such as chronic lung disease, cyanotic congenital heart disease, neuromuscular disease, a primary immunodeficiency, prematurity (<36 weeks) or had respiratory distress unrelated to a viral URI. This study was approved by the Institutional Review Board (IRB) of Human Subject Research at Baylor College of Medicine and Affiliated Institutions which includes Texas’s Children Hospital. The IRB is registered with the Office for Human Research Protections of the U.S. Department of Health and Human Services. All personal identifying information have been removed from the data set. <br> <p> </p

Demographic and clinical characteristics by disposition.

<p>Demographic and clinical characteristics by disposition.</p

Description of the RNase P-normalized copy numbers of 5 RSV genes.

<p>Description of the RNase P-normalized copy numbers of 5 RSV genes.</p

RNase P-normalized RSV gene copy numbers between non-hospitalized and hospitalized infants.

<p>RNase P-normalized RSV gene copy numbers between non-hospitalized and hospitalized infants.</p

FLN_GR_F07_narrativa_cam02

<p>Genotype specific amino acid changes when compared to the RSV/A Long strain in antigenic site ø.</p

Spearman correlations among the RNase P-normalized copy numbers of 5 RSV genes.

<p>Spearman correlations among the RNase P-normalized copy numbers of 5 RSV genes.</p

Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B

<div><p>Background</p><p>The fusion (F) protein of RSV is the major vaccine target. This protein undergoes a conformational change from pre-fusion to post-fusion. Both conformations share antigenic sites II and IV. Pre-fusion F has unique antigenic sites p27, ø, α2α3β3β4, and MPE8; whereas, post-fusion F has unique antigenic site I. Our objective was to determine the antigenic variability for RSV/A and RSV/B isolates from contemporary and historical genotypes compared to a historical RSV/A strain.</p><p>Methods</p><p>The F sequences of isolates from GenBank, Houston, and Chile (N = 1,090) were used for this analysis. Sequences were compared pair-wise to a reference sequence, a historical RSV/A <i>Long</i> strain. Variability (calculated as %) was defined as changes at each amino acid (aa) position when compared to the reference sequence. Only aa at antigenic sites with variability ≥5% were reported.</p><p>Results</p><p>A total of 1,090 sequences (822 RSV/A and 268 RSV/B) were analyzed. When compared to the reference F, those domains with the greatest number of non-synonymous changes included the signal peptide, p27, heptad repeat domain 2, antigenic site ø, and the transmembrane domain. RSV/A subgroup had 7 aa changes in the antigenic sites: site I (N = 1), II (N = 1), p27 (N = 4), α2α3β3β4(AM14) (N = 1), ranging in frequency from 7–91%. In comparison, RSV/B had 19 aa changes in antigenic sites: I (N = 3), II (N = 1), p27 (N = 9), ø (N = 4), α2α3β3β4(AM14) (N = 1), and MPE8 (N = 1), ranging in frequency from 79–100%.</p><p>Discussion</p><p>Although antigenic sites of RSV F are generally well conserved, differences are observed when comparing the two subgroups to the reference RSV/A <i>Long</i> strain. Further, these discrepancies are accented in the antigenic sites in pre-fusion F of RSV/B isolates, often occurring with a frequency of 100%. This could be of importance if a monovalent F protein from the historical GA1 genotype of RSV/A is used for vaccine development.</p></div