www.nature.com/pr BASIC SCIENCE ARTICLE OPEN Gene signature of children with severe respiratory syncytial virus infection Clyde Dapat 1, Satoru Kumaki2, Hiroki Sakurai3, Hidekazu Nishimura4, Hannah Karen Mina Labayo1, Michiko Okamoto1, Mayuko Saito1 and Hitoshi Oshitani1 BACKGROUND: The limited treatment options for children with severe respiratory syncytial virus (RSV) infection highlights the need for a comprehensive understanding of the host cellular response during infection. We aimed to identify host genes that are associated with severe RSV disease and to identify drugs that can be repurposed for the treatment of severe RSV infection. METHODS: We examined clinical data and blood samples from 37 hospitalized children (29 mild and 8 severe) with RSV infection. We tested RNA from blood samples using next-generation sequencing to profile global mRNA expression and identify cellular processes. RESULTS: Retractions, decreased breath sounds, and tachypnea were associated with disease severity. We observed upregulation of genes related to neutrophil, inflammatory response, blood coagulation, and downregulation of genes related to T cell response in children with severe RSV. Using network-based approach, 43 drugs were identified that are predicted to interact with the gene products of these differentially expressed genes. CONCLUSIONS: These results suggest that the changes in the expression pattern in the innate and adaptive immune responses may be associated with RSV clinical severity. Compounds that target these cellular processes can be repositioned as candidate 1234567890();,: drugs in the treatment of severe RSV. Pediatric Research (2021) 89:1664–1672; https://doi.org/10.1038/s41390-020-01347-9 IMPACT: ● Neutrophil, inflammation, and blood coagulation genes are upregulated in children with severe RSV infection. ● Expression of T cell response genes are suppressed in cases of severe RSV. ● Genes identified in this study can contribute in understanding the pathogenesis of RSV disease severity. ● Drugs that target cellular processes associated with severe RSV can be repositioned as potential therapeutic options. INTRODUCTION have been conducted using high throughput technologies to Acute respiratory tract infections due to viral pathogens are the identify host factors that are affected during RSV infection.3–5 major causes of illness and death in children worldwide. The However, these studies utilized in vitro cell lines and laboratory- leading cause of acute lower respiratory tract infection among adapted strains of RSV, which may not reflect the clinical course of children younger than 5 years of age is respiratory syncytial virus the disease. Studies were conducted using whole blood from (RSV), which affects 33.1 million children and responsible for 3.2 children with acute respiratory infections to distinguish between million hospital admissions and 59,600 in-hospital deaths per year mild and severe forms of pneumonia.6–9 These studies utilized worldwide.1 Almost all children will get infected with RSV in their various case definitions of severe RSV, which may be challenging first year of life that is characterized by a spectrum of infection to implement across different settings. The World Health from mild respiratory symptoms to severe pneumonia and Organization (WHO) working group proposed a case definition bronchiolitis, which affects 3.4 million children worldwide.2 for severe and very severe RSV-associated lower respiratory tract Children with severe RSV require hospitalization and some would illness (LRTI), which is based on clinical features that are objective need intensive care. and easy to understand including cough, difficulty breathing, and 10 To date, vaccine against RSV is under development and the SpO2 measurements. treatment option for children with severe RSV is limited. This may Combining genomic data with clinical information may provide be due to our incomplete understanding of the host cellular insight into the development of RSV disease severity in children. responses during RSV infection. One of the major difficulties in This study aimed to identify host genes in children that are providing host-directed therapy is the lack of reliable diagnostic associated with severe RSV-associated lower respiratory tract tools in the management of severe RSV. Several in vitro studies infection using the WHO case definition. This study also aimed to 1Department of Virology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan; 2Department of Pediatrics, Sendai Medical Center, 11-12 Miyagino 2-chome, Miyagino-ku, Sendai 983-8520, Japan; 3Department of General Pediatrics, Miyagi Children’s Hospital, 3-17 Ochiai 4-chome, Aoba-ku, Sendai 989-3126, Japan and 4Virus Research Center, Sendai Medical Center, 11-12 Miyagino 2-chome, Miyagino-ku, Sendai 983-8520, Japan Correspondence: Clyde Dapat ([email protected]) Received: 27 August 2020 Revised: 15 November 2020 Accepted: 14 December 2020 Published online: 28 January 2021 © The Author(s) 2021 Gene signature of children with severe respiratory syncytial virus. C Dapat et al. 1665 identify host genes that codes for proteins as potential drug (Illumina, San Diego, CA). Libraries were sequenced using the targets, which will interact with drugs as possible treatment Illumina Hiseq 2500 (Illumina, San Diego, CA) at a target depth of options for severe RSV infection. about 20 million 51-nucleotide single-end reads per sample. Differential gene expression analysis METHODS Next-generation sequencing (NGS) data were analyzed using the Study design, setting, and participants SeqBox system.13 Briefly, raw reads were trimmed using skewer,14 This was a prospective observational study of hospitalized children mapped to the hg38 human reference genome using STAR,15 and infected with RSV evaluating the host genes that were associated quantified with RSEM.16 PCAExplorer R package version 2.14.2 with severe disease. We enrolled children younger than 5 years (ref. 17) was used for principal component analysis and heatmap old with coughing, sneezing, nasal discharge, wheezing, or visualizations on normalized and log-transformed sequence data breathing difficulties admitted at Sendai Medical Center and by the variance stabilizing transformation method.18 Differential Miyagi Children’s Hospital in Sendai, Japan during the two RSV expression analysis was performed on un-normalized sequence seasons from October 2017 to March 2019. We excluded children count data by comparing the two groups (mild vs. severe RSV) with known heart, lung, liver, or kidney disease, immunodefi- after adjusting for age, sex, and batch by enrollment year and ciency, gestational age <36 weeks, received steroid treatment hospital using DESeq2 R package version 1.28.1.19 Results of within 2 weeks or palivizumab treatment within 4 weeks. This DESeq2 analysis included p values, adjusted p values, and log2 study was approved by the Tohoku University Graduate School of fold changes with positive log fold change values indicate Medicine Ethics Committee, Sendai Medical Center Ethics increased gene expression while negative values indicate Committee, and Miyagi Children’s Hospital Ethics Committee. decreased gene expression in the severe RSV group when Written informed consent was obtained from the parent or compared to mild RSV group. The empirical Bayes method was guardian before participating in the study. used to adjust for nonbiological variation between batches as implemented in the surrogate variable analysis (SVA) R package Data and sample collection version 3.36.0.20 False discovery rate (FDR) was calculated by Demographic and clinical information were collected upon applying the weighted Benjamin–Hochberg method for multiple admission using questionnaires and medical records. Nasal hypothesis testing using the independent hypothesis weighting samples (nasopharyngeal swabs, nasal aspirate, or nasal wash) (IHW) software.21 A gene was considered differentially expressed if were collected upon admission and tested for RSV by the rapid its FDR < 0.05. NGS data are deposited in the NCBI Gene diagnostic test kit. Peripheral whole blood (1 mL) was collected Expression Omnibus (GEO accession number: GSE155925). upon admission using PAXgene Blood RNA tubes (PreAnalytix GmbH, Hombrechtikon, Switzerland). Other clinical data including Functional enrichment analysis length of hospital stay, oxygen administration, chest X-ray, and To determine the biological function of differentially expressed blood count were collected after hospital discharge. genes, modular transcriptome analysis was performed using the tmod R package version 0.44.22 GO database release version 2020- Assessment of RSV severity 07 (refs. 23,24) and KEGG database release version 95.0 (ref. 25) were Severity of the disease was assessed based on the WHO case used in the functional enrichment analysis. definition for severe and very severe RSV-associated lower respiratory tract infection.10 Respiratory tract illness (RTI) was Gene network analysis defined as having cough and/or difficult breathing. LRTI was Gene co-expression network was generated by querying the list of defined as having RTI (cough or difficulty breathing) and differentially expressed genes to the STRING database version 11.0 tachypnea (≥60 breaths per min for children <2 months, ≥50 (ref. 26). Genes that are expressed only in blood were included in breaths per min for children 2–11 months, and ≥40 breaths per the analysis to minimize false positives. The reconstructed