Recurrent rhinovirus infections in a child with inherited MDA5 deficiency

Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.

rnA-Seq A549 cells that had been transfected 48 h earlier with siRNA to MDA5 or nonspecific negative control siRNA, were uninfected or infected for 6, 12, 24, and 48 h with HRV-B14 or RSV, as described in the RSV infections sec-tion. For each time point, infections were performed in triplicate. Total cellular RNA were isolated using RNeasy Mini kit (QIA GEN). Multiplexed RNA libraries were prepared using the Truseq RNA sample prep kit (Illumina). In brief, poly(A)-containing mRNA were captured with oligodT beads, fragmented, reverse-transcribed, and the cDNA was ligated to Illumina adapters containing indexing barcodes. Libraries were quantified using KAPA Library Quant kits (KAPA Biosystems), before running on a HiSeq 2000 Sequencing System (Illumina) to produce 50 bp single end reads. Sequencing reads were aligned with ELA ND to the human reference genome version hg19. Count data of the annotated transcripts from individual samples were normalized for sequence depth. Analyses of differentially expressed genes were performed using the Statistical R package DESeq2. Sequencing reads were also aligned with Bowtie2 to the HRV-B14 or RSV reference genomes to count the read number corresponding to the virus transcripts. The RNA-seq coverage across the virus transcripts were checked for normal distribution and count data of the virus transcripts were also normalized for overall sequence depth. Fold changes were calculated by comparing the RPKM expression values (Reads Per Kilobase per Million mapped reads) under siNeg vs. siMDA5 conditions. For human genes, log 2 -transformed (base 2) expression fold changes from DESeq2 output were analyzed for distribution (box-and-whisker diagrams) and hierarchical clustering (heat map of expression values) using R. "Response to type I interferon" genes were selected based on the GO term definition (GO term GO0034340). The comparisons for the fold change distributions of different gene sets were performed using two sample Kolmogorov-Smirnov tests (KS tests). RNA-seq data were deposited into the NCBI BioProject database and the Sequence Read Archive (SRA) under accession no. PRJ NA387035.