RNA sequencing and pathway enrichment data analysis
RNA from the whole blood of 16 MHV-1- and 16 mock-infected C3H/HeJ mice were submitted to BGI (Cambridge, MA, USA) to perform library prep and sequencing using their DNBseq platform. RNA quality was assessed using Nanodrop 260/280 and 260/230 ratios and raw reads were generated as Fastq files. We performed quality control on the raw reads using FastQC26 and trimmed them using BBDuk27. The mouse reference genome (GRCm39) and corresponding annotations were downloaded from National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). The clean reads were mapped to the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) with default parameters28. HT-Seq (htseq-count) within the STAR package was used to estimate gene counts29. Differential gene expression analysis was conducted using DESeq230, and Gene Set Enrichment Analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. We employed the integrated differential expression and pathway analysis tool (iDEP 0.96, South Dakota University, SD, USA)31 for these analyses, setting the default parameters to a p-adjusted value of <0.1 and fold change of >2 for identifying differentially expressed genes. The expression patterns of identified biomarker genes were plotted with gene plot feature in iDEP 2.0 using raw counts and standard deviation.