Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection

Background: Respiratory syncytial virus (RSV) is the major viral driver of a global pediatric respiratory disease burden disproportionately borne by the poor1. Thus, RSV, like SARS-CoV-2, combines with congenital and environmental and host-history-dependent factors to create a spectrum of disease with greatest severity most frequently occurring in those least able to procure treatment. Methods: Here we apply whole genome sequencing and a suite of other molecular biological techniques to survey host-virus dynamics in infections of two distinct cell lines (HEp2 and A549) with four strains representative of known RSV genetic diversity. Results: We observed non-gradient patterns of RSV gene expression and a single major difference in transcriptional readthrough correlating with a deep split in the RSV phylogenetic tree. We also observed increased viral replication in HEp2 cells along with a pro-inflammatory host-response; and decreased viral replication in A549 cells with a more potent antiviral response in host gene expression and levels of secreted cytokines. Conclusions: Our findings suggest HEp2 and A549 cell lines can be used as complementary models of host response leading to more or less severe RSV disease. In vitro perturbations inspired by actual environmental and host-history-dependent factors associated with greater disease can be tested for their ability to shift the antiviral response of A549 cells to the more pro-inflammatory response of HEp2 cells. Such studies would help illuminate the tragic costs of poverty and suggest public health-level interventions to reduce the global disease burden from RSV and other respiratory viruses

Antigenic Site-Specific Competitive Antibody Responses to the Fusion Protein of Respiratory Syncytial Virus Were Associated With Viral Clearance in Hematopoietic Cell Transplantation Adults

Background: Recent studies of human sera showed that the majority of the respiratory syncytial virus (RSV) neutralizing antibodies are directed against pre-fusion conformation of the fusion (F) protein of RSV and revealed the importance of pre-fusion antigenic site Ø specific antibodies. However, detailed analysis of multiple antigenic site-specific competitive antibody responses to RSV F protein and their contribution to virus clearance in humans are lacking.Methods: We prospectively enrolled a cohort of RSV infected hematopoietic cell transplantation (HCT) adults (n = 40). Serum samples were collected at enrollment (acute, n = 40) and 14 to 60 days post-enrollment (convalescent, n = 40). Antigenic site-specific F protein antibodies were measured against pre-fusion site Ø, post-fusion site I, and sites II and IV present in both the pre-fusion and post-fusion F protein conformations utilizing four different competitive antibody assays developed with biotinylated monoclonal antibodies (mAb) D25, 131-2A, palivizumab, and 101F, respectively. The lower limit of detection were 7.8 and 1.0 μg/mL for the competitive antibody assays that measured site Ø specific response, as well as sites I, II, and IV specific responses, respectively. Neutralizing antibody titers to RSV A and B subgroups was determined by microneutralization assays.Results: The overall findings in RSV infected HCT adults revealed: (1) a significant increase in antigenic site-specific competitive antibodies in convalescent sera except for site Ø competitive antibody (p < 0.01); (2) comparable concentrations in the acute and convalescent serum samples of antigenic site-specific competitive antibodies between RSV/A and RSV/B infected HCT adults (p > 0.05); (3) significantly increased concentrations of the antigenic site-specific competitive antibodies in HCT adults who had genomic RSV detected in the upper respiratory tract for <14 days compared to those for ≥14 days (p < 0.01); and (4) statistically significant correlation between the antigenic site-specific competitive antibody concentrations and neutralizing antibody titers against RSV/A and RSV/B (r ranged from 0.33 to 0.83 for acute sera, and 0.50–0.88 for convalescent sera; p < 0.05).Conclusions: In RSV infected HCT adults, antigenic site-specific antibody responses were induced against multiple antigenic sites found in both the pre-fusion and post-fusion F conformations, and were associated with a more rapid viral clearance and neutralizing antibody activity. However, the association is not necessarily the cause and the consequence

Gene sequence variability of the three surface proteins of human respiratory syncytial virus (HRSV) in Texas.

Human respiratory syncytial virus (HRSV) has three surface glycoproteins: small hydrophobic (SH), attachment (G) and fusion (F), encoded by three consecutive genes (SH-G-F). A 270-nt fragment of the G gene is used to genotype HRSV isolates. This study genotyped and investigated the variability of the gene and amino acid sequences of the three surface proteins of HRSV strains collected from 1987 to 2005 from one center. Sixty original clinical isolates and 5 prototype strains were analyzed. Sequences containing SH, F and G genes were generated, and multiple alignments and phylogenetic trees were analyzed. Genetic variability by protein domains comparing virus genotypes was assessed. Complete sequences of the SH-G-F genes were obtained for all 65 samples: HRSV-A = 35; HRSV-B = 30. In group A strains, genotypes GA5 and GA2 were predominant. For HRSV-B strains, the genotype GB4 was predominant from 1992 to 1994 and only genotype BA viruses were detected in 2004-2005. Different genetic variability at nucleotide level was detected between the genes, with G gene being the most variable and the highest variability detected in the 270-nt G fragment that is frequently used to genotype the virus. High variability (>10%) was also detected in the signal peptide and transmembrane domains of the F gene of HRSV A strains. Variability among the HRSV strains resulting in non-synonymous changes was detected in hypervariable domains of G protein, the signal peptide of the F protein, a not previously defined domain in the F protein, and the antigenic site Ø in the pre-fusion F. Divergent trends were observed between HRSV -A and -B groups for some functional domains. A diverse population of HRSV -A and -B genotypes circulated in Houston during an 18 year period. We hypothesize that diverse sequence variation of the surface protein genes provide HRSV strains a survival advantage in a partially immune-protected community

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

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

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

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

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

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