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

FLN_GR_F07_narrativa_cam02

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

Genotype specific amino acid changes when compared to the RSV/A <i>Long</i> strain in antigenic site MPE8.

<p>Genotype specific amino acid changes when compared to the RSV/A <i>Long</i> strain in antigenic site MPE8.</p

The entropy values in amino acids within antigenic sites of the fusion gene of RSV/A and RSV/B have a similar distribution.

<p>Entropy was defined as ∑_<i>i</i>−<i>i</i> log(<i>i</i>). Individual genotypes of each subgroup contributed equally to the proportion of amino acids found at a given residue.</p

Frequency of amino acid changes in antigenic sites for of the fusion gene RSV/A (N = 822) and RSV/B (N = 268) compared to the RSV/A <i>Long</i> strain.

<p>Frequency of amino acid changes in antigenic sites for of the fusion gene RSV/A (N = 822) and RSV/B (N = 268) compared to the RSV/A <i>Long</i> strain.</p

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

Amino acids of the RSV fusion gene with the greatest 5% entropy among isolates in RSV/A and RSV/B subgroups.

<p>Amino acids of the RSV fusion gene with the greatest 5% entropy among isolates in RSV/A and RSV/B subgroups.</p

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

The fusion genes of RSV/A isolates are more similar to the RSV/A <i>Long</i> strain than RSV/B isolates.

<p>Non-synonymous/synonymous ratio graph of amino acids with non-synonymous or synonomous changes in the fusion gene for a) RSV/A isolates and b) RSV/B isolates (compared to the RSV/A <i>Long</i> strain). Fusion gene domains are depicted by assigned color blocks. Antigenic sites are highlighted in shades of gray respective to the protein conformation on which they are found.</p

Appearance of RSV/A and RSV/B genotypes and dominance over time (1961–2014).

<p>Sequences assigned genotypes were assessed by their sample acquisition date. The included inset depicts those years (1961–2000) with a small number of available sequences.</p