miRNAs differentially expressed during virus infection. (a) Overview of 45 miRNAs differentially expressed in mouse lung samples during SARS-CoV (MA15) or influenza virus (PR8) infection. Colors on the heat map indicate the log₂ ratios of expression (representing normalized read counts) in virus-infected samples to expression in matched mock-infected samples. Red, upregulation; green, downregulation. The data in the eight columns under “small RNA NGS” represent log₂ ratios of expression in virus-infected samples to expression in mock-infected samples as determined on the basis of small RNA transcriptome sequencing analysis. NGS, next-generation sequencing. The nine columns under “published microarray” represent log₂ ratios determined on the basis of the microarray measurements of the corresponding miRNAs in lung samples from a previous study (3) in which mice were infected with the fully reconstructed 1918 pandemic (1918) or a nonlethal seasonal (Tx) influenza virus. The three columns under “1918/Tx” show the results of direct comparisons of miRNA expression in 1918-infected samples to expression in Tx-infected samples at 1, 3, and 5 days after infection; note that only changes of at least 1.5-fold are indicated. The log₂ ratios of expression in infected samples to expression in mock-infected samples are shown separately under “1918” and “Tx,” where rows in grey indicate that the corresponding miRNAs were not probed by the miRNA microarray and rows in white indicate undetected miRNAs. The left sidebar shows the relative abundances of the corresponding miRNAs represented as the minimum average normalized read counts across all samples. (b) Patterns of changes in expression of selected miRNAs across a panel of virus infection samples measured by qPCR. Six miRNAs from panel a (indicated in boldface and italics) were selected; two, namely, miR-155 in macrophages (10) and miR-223 in neutrophils (11), are known to be related to immune cells. The other four miRNAs came from a small-scale screening performed using qPCR with a subset of randomly selected differentially expressed miRNAs in PWK/PhJ mouse embryonic fibroblasts (MEFs) infected with influenza virus (data not shown). Also, miR-27a* and miR-671-3p were not measured by previous microarray (see panel a). Colors on the heat map indicate the log₂ ratios (the differences between medians of normalized Ct values of replicate samples) of expression in virus-infected samples to expression in matched mock-infected samples. Red, upregulation; green, downregulation. The “Lung + Flu” data contrast miRNA expression changes in lung samples from mice infected with highly pathogenic influenza virus (WT, VN1203) and with minimally pathogenic influenza virus (HA, VN1203 with a mutation in HA protein) at two time points: days 2 and 4 after infections. Similarly, the “Lung + SARS” data contrast miRNA expression changes in lung samples from mice infected with highly pathogenic SARS-CoV (MA, MA15) and minimally pathogenic SARS-CoV (Urb, Urbani). The “PWK MEFs + Flu” data represent temporal miRNA expression changes in samples from cultured PWK MEFs infected with the mouse-adapted A/PR/8/34 influenza virus across four time points after infection. A more detailed representation of individual replicate experiments is shown in Fig. S3. WT, wild type.

Examples of two nonannotated small RNAs. (a) An overview of a 400-bp region of chromosome 5 similar to that shown in Fig. 3b. The predicted small RNA is located in an intron of the Trim56 protein-coding gene (top). Both Trim56 and the predicted small RNA are on the negative strand. (b) An overview of a 400-bp region of chromosome X similar to that shown in panel a. The predicted small RNA is antisense to an intronic region of the Il1rapl1 protein-coding gene (top). Ll1rapl1 is on the negative strand, and the predicted small RNA is on the positive strand.

Overview of deep-sequencing analysis of the small RNA transcriptome in lungs from virus-infected mice. (a) Length distribution of small RNA short reads in an arbitrarily chosen sample. Reads were adaptor trimmed. Reads < 16 nt were discarded, and reads > 40 nt were shortened to 40 nt by clipping from the 3 ′ end. (b) Global classification of small RNA transcriptional activity of the sample described in the panel a legend. Short reads were sequentially mapped to sequences of ribosomal RNAs (rRNA), transfer RNAs (tRNA), small cytoplasmic RNAs, small nuclear RNAs, small nucleolar RNAs (other ncRNAs), piwi-associated small RNAs (piRNA), microRNA precursor sequences, a mouse reference genome, and the SARS-CoV genome. (c) Relative abundances of the most abundant mature miRNAs in lung samples from virus-infected mice. The 10 most abundant mature miRNAs in at least 4 samples across all 20 samples are shown. (d) Distribution of detected mature miRNAs grouped by normalized read count (the number of the reads per million mapped reads) across all 20 samples. Numbers in parentheses represent ranges. SARS, SARS-CoV; FLU, influenza virus.

Length distributions of identified putative small RNA loci and two examples of non-miRNA small RNAs differentially expressed during SARS-CoV and influenza virus infection. (a) The length distribution of the identified putative small RNA loci. Data for “overlap annotated smRNAs” represent loci overlapping annotated or predicted small RNAs, including miRNAs, snoRNAs, piRNAs, and RNA repeats. “No overlap” data represent loci that do not overlap any small RNAs. (b) UCSC genome browser (http://genome.ucsc.edu) display of the locations of reads mapped to a 350-bp region of chromosome 6 (chr6). On the left, each track represents short reads collected from small RNA transcriptome sequencing analysis of a single mouse lung sample. Because there were too many reads to be displayed individually, total reads were condensed (dense mode) to display the genomic regions with mapped reads. The red color indicates that the reads were mapped to the negative strand. The black horizontal bar under the sample tracks shows the location of the predicted small RNA locus. The red horizontal bar represents a Y RNA, a small noncoding RNA component of the Ro ribonucleoprotein particle, as annotated by the use of Ensembl software. The raw total read counts of the corresponding small RNA locus across all 20 samples are represented on the right in the same order as shown on the left. Read counts indicate that expression of the small RNA was upregulated during virus infection. Mammal cons, UCSC track (http://genome.ucsc.edu) showing the placental mammal basewise conservation by PhyloP. (c) An overview similar to that shown in panel b for a 700-bp region of chromosome 1 covering two predicted small RNA loci. Each track represents a condensation of the short reads from a single sample (middle panel). The blue color indicates the reads were mapped to the positive strand. Green boxes show two predicted small RNA loci overlapped with two snoRNAs (the red horizontal bar inside each green box) annotated by the use of Ensembl software (version 61) and located in the introns of Gas5, a known long ncRNA located on the positive strand. Different isoforms of Gas5 are shown on the track labeled Ensembl Genes. An expanded view of the read mapping highlighted by the red box is displayed on the left. Distributions of the lengths and mapped locations of short reads from the single sample are shown. Raw read counts of two corresponding small RNA loci across all 20 samples are shown on the right (in the same order as shown in the middle panel). Both small RNA loci were upregulated during SARS-CoV and influenza virus infection.

Correlations of mature miRNA expression changes with the corresponding pri-miRNA estimates. (a) Scatterplot of the log₂ fold changes of mature miRNA expression measured by small RNA transcriptome sequencing analysis (y axis) and the log₂ fold changes in expression of the annotated loci overlapped with the corresponding miRNAs measured by whole-transcriptome sequencing analysis using the same samples (x axis). In total, 36 mature miRNA loci overlapped annotated loci and were included in the plot. (b) Data are presented as described for panel a, with the y axis data showing the log₂ fold changes in expression of the 2-kb genomic regions surrounding the corresponding miRNA precursors measured by whole-transcriptome sequencing analysis of the same samples. In total, 42 mature miRNAs were included in the plot, as 3 of 45 genomic regions surrounding differentially expressed miRNAs were not subjected to differential expression analysis due to the lack of a sufficient number of reads for quantification in the whole-transcriptome sequencing data.