Fig 1.
Induction of cytokines and chemokines by clinical isolates of RSV in A549 cells and primary monocyte-derived human macrophages (MDM).
(A) A549 cells or (B) MDM (donor #66, 67 and 68) were infected with clinical isolates NH1125B or NH1067B. Mock infected cells were used as controls. At multiple time points post-infection, cell culture supernatant were collected and concentration of IL-6 and RANTES (CCL5) were measured by BioPlex assay. For the A549 cell experiments, biological triplicates were used and the error bars are displayed (for several time points, the error bars are too small to be seen in the figure). To make the MDM data from different donors compatible, we applied a standardization procedure with the average expression over all time points (for each donor) equal zero and the standard deviation equal 1. Data along ordinate axis is given in these standardized units. Nineteen donors (including donor #66, 67 and 68) were screened for secretion of IL-6 (C) and CCL5 (D) at 24 hours post infection using the methodologies described above.
Fig 2.
Transcriptome analyses of A549 cells and MDM during infection with clinical isolates of RSV.
A549 cells and MDM (donor#64) were infected with NH1067B and NH1125B. RNA was extracted at the designated times post infection and gene expression was determined by RNA-SEQ (A) or quantitative RT-PCR (B). (A) Heat map illustrating changes in cytokine and chemokine transcription following infection. Individual genes are in rows and variations in expression are depicted using the color scale on the right of the figure. Each column is a time point (in hours) post infection. “U” represents mock infected cells. Each row represents a unique cytokine. Arrows point to transcription of IL-6, CCL5 and TNFα. (B) Quantitative RT-PCR of specific genes in virus infected A549 and MDMs. RNA was extracted from MDM from donor #67 and 68 at several times post infection (as described above) and gene expression was determined by quantitative RT-PCR. The Y-axis represents fold expression over mock infected cells and error bars are included for statistical perspectives.
Fig 3.
Host gene expression in response to RSV infection.
A549 cells or MDM (donor #64) were infected with NH1067B and NH1125B. RNA was extracted at the designated times post infection and gene expression was determined by RNA-SEQ. Individual genes are in rows and variations in expression are depicted using the color scale on the right of the figure. Expression of genes favorable for virus infection (viral entry and viral replication) and expression of genes in response to viral infection (host-viral recognition, innate immunity response, interferon (IFN) response and adaptive immunity) are clustered and relative expression induced by each virus is presented. Some of the genes and biological and biochemical pathways assessed are listed in each category. Mock infected (U) cells were used as controls.
Fig 4.
Comparison of time dynamics of cellular genes in response to infection with RSV NH1067B or NH1125B.
MDM (donor #64) (A) or A549 cells (B) were infected with NH1067B and NH1125B. RNA was extracted at the designated times post infection and assayed by RNA-SEQ. Time dynamics of expression of genes favorable for virus infection (viral entry and viral replication) and the innate immune response to viral infection (host-viral recognition, innate immunity response, interferon (IFN) response and adaptive immunity) are shown separately in each graph. Representative genes are labeled.
Fig 5.
Host-associated differences in response to RSV infection.
MDM obtained from 5 donors (#40, #64, #52, #54, #66) were infected with NH1125B. At 24 hours post infection RNA was extracted and gene expression profiles were determined by RNA-SEQ. This heatmap includes expression of genes supporting virus infections (viral entry and replication; see Fig 3) and expression of genes in response to viral infection (host-viral recognition, innate immunity, interferon response and adaptive immunity; see Fig 3).
Fig 6.
Functional fingerprint of cellular gene expression in MDM infected with multiple clinical isolates of RSV subtype B.
(A) Transcriptional heatmaps of cellular genes of MDM (donor #66) infected with RSV. RNA was extracted 18 hours post infection and gene expression was determined by RNA-SEQ. Each row represents a virus (18 hours post infection) or mock infected cells (M) processed at 0 or 18 hours. Clinical isolates include subtype B strains NH1125B (1125), NH1001B (1001), NH1161B (1161), NH1067B (1067), NH1182B (1182), and TX11-56B (11–56). Viruses were designated as “low” or “high” inducers based on their capacity to induce IL-6 and RANTES as shown in the graph (B). IL-6 and RANTES secretion in response to viral infection was measured by BioPlex. (C) Heatmap demonstrating the expression of a subset of cellular genes in MDM infected with clinical isolates of RSV. These genes were selected based on published data indicating that their expression can be regulated by the RSV G protein and or interferon-stimulated gene.
Fig 7.
Phylogeny of RSV isolates and correlation to the induction phenotype within the viral genome.
(A) Phylogenetic analysis of 33 RSV A and B strains. Analysis was based on the whole genome sequence of each virus. The portion of the dendrogram circle in pink represents B strains and the portion of the dendrogram circle in light blue represents A strains. “High inducers” are in red text and “low inducers” are in blue text. (B) Phylogenic analysis based on the region of the viral genome containing the sequential genes SH, G and F. Sequence and phylogenic analysis suggested that the induction phenotype (“high” vs. “low”; see Fig 6) mapped to this region of the genome. Based on these analyses, segregation of the viral isolates correlated with the cytokine/chemokine induction phenotype.
Fig 8.
Sequence analyses of RSV isolates and correlation to the induction phenotype within the viral genome.
(A) Amino acid sequence of a portion of the G protein of clinical isolates of RSV B. Amino acid sequence, consensus sequence and amino acid number are displayed. The G gene of “low inducers” contains a 20 amino acid duplication (corresponding to the 60 base duplication) absent in “high inducers”. Like the duplication, amino acid residue at position 229 corresponds to viral induction phenotype (isoleucine in “low inducers” and threonine in “high inducers”). (B) Amino acid sequence of a portion of the M2-1 protein of clinical isolates of RSV B. Amino acid sequence, consensus sequence and amino acid number are displayed. Amino acid residue at position 142 corresponds to viral induction phenotype (serine in “low inducers” and asparagine in “high inducers”).
Fig 9.
Pathway analysis of interferon stimulated genes after RSV infection.
Interferon stimulated genes are organized based on known established pathways and subcellular locations. Symbols are bi-colored with the left half characterizing genes activated by NH1125B and the right half genes activated by NH1067B. The differences in color intensity reflect differences in the expression levels of the genes by each virus. Data for this figure were obtained at 8 hours (A) and 24 hours (B) post infection of MDM cells.
Table 1.
Genes associated with RSV pathogenesis and expressed differently in response to NH1125B or NH1067B infection.
Table 2.
List of genes up-regulated in high inducers infection vs. low inducers and were identified in Reference 64 as up-regulated in blood cells in RSV infected infants (relative to control).