Data from the 24 PCMV clinical isolates and prototype Sichuan strain (GenBank accession no. JN701021.1).

<p>Strains are designated according to accession number and strain name.</p

Transcriptome Analysis of Porcine Thymus following Porcine Cytomegalovirus Infection

<div><p>Porcine cytomegalovirus (PCMV) is a major immunosuppressive virus that mainly affects the immune function of T lymphocytes and macrophages. Despite being widely distributed around the world, no significantly different PCMV serotypes have been found. Moreover, the molecular immunosuppressive mechanisms of PCMV, along with the host antiviral mechanisms, are still not well characterized. To understand the potential impact of PCMV on the function of immune organs, we examined the transcriptome of PCMV-infected thymuses by microarray analysis. We identified 5,582 genes that were differentially expressed as a result of PCMV infection. Of these, 2,161 were upregulated and 3,421 were downregulated compared with the uninfected group. We confirmed the expression of 13 differentially expressed immune-related genes using quantitative real-time RT-PCR, and further confirmed the expression of six of those cytokines by western blot. Gene ontology, gene interaction networks, and KEGG pathway analysis of our results indicated that PCMV regulates multiple functional pathways, including the immune system, cellular and metabolic processes, networks of cytokine-cytokine receptor interactions, the TGF-β signaling pathway, the lymphocyte receptor signaling pathway, and the TNF-α signaling pathway. Our study is the first comprehensive attempt to explore the host transcriptional response to PCMV infection in the porcine immune system. It provides new insights into the immunosuppressive molecular mechanisms and pathogenesis of PCMV. This previously unrecognized endogenous antiviral mechanism has implications for the development of host-directed strategies for the prevention and treatment of immunosuppressive viral diseases.</p></div

Length distribution of total sRNAs in TGEV-infected ST cells and normal ST cells.

<p>(A) Bar chart showing the total read counts against the read lengths for the complete adapter-trimmed read set in TGEV-infected ST cells. (B) Bar chart shows the total read counts against the read lengths for the complete adapter-trimmed read set in normal ST cells. The results indicate a successful enrichment of mature miRNAs in the TGEV-infected ST cells and normal ST cell libraries.</p

Scatter plot of the high-throughput sequencing data.

<p>The high-throughput sequencing data (differentially expressed miRNAs) are graphed on the scatter plot to visualize variations in miRNA expression between TGEV infected and control groups. The values on the X axes and Y axes of the scatter plot are the normalized values for the TGEV infected and control groups (log2 scaled). The green lines are fold-change lines (default fold-change value: 1.5).</p

Plots of transition (s) and transversion (v) frequencies against the K80 distance.

<p><i>S</i> denotes transition and <i>V</i> denotes transversion. (A) Analysis of 34 PCMV strains from China. (B) Analysis of global PCMV strains.</p

Pathological section examination.

<p>A–E: Control porcine (A) lung, (B) liver, (C) spleen, (D) kidney, and (E) thymus tissue sections. F–J: PCMV-infected porcine (F) lung, (G) liver, (H) spleen, (I) kidney, and (J) thymus tissue sections.</p

Phylogenetic analysis of 42 global strains of PCMV based on the 2580 bp <i>gB</i> complete nucleotide sequence and the deduced amino acid sequence.

<p>(A) Phylogenetic tree constructed from nucleotide sequences. (B) Phylogenetic tree constructed from amino acid sequences. The reference PCMV <i>gB</i> nucleotide sequences were obtained from GenBank: SC strain (accession no. JN701021.1); ZZ strain (accession no. FJ870562.1); NB strain (accession no. FJ844360.1, FJ870561.1); FJ strain (accession no. FJ870564.1); JH strain (accession no. FJ870563.1); HN isolate (accession no.HQ686081.1, HQ686080.1, FJ595497.1, EF460488.1); Spanish 55b isolate (accession no. AF268040.2); B6 strain (accession no. AF268039.2); Japanese OF-1 strain (accession no. AF268041.2); Japanese Yamaguchi strain (accession no. AB771707.1, AB771708.1, AB771706.1); Swedish isolate P1 (accession no. AF394057.1); Swedish isolate 1469 (accession no. AF394056).Multiple alignment was performed in the Clustal W program, and MEGA 5.0 software was used to construct the neighbor-joining (NJ) and maximum likelihood (ML) trees. Bootstrap values >65% from 1000 pseudo-replicates are indicated at the branch nodes. Numbers in parentheses are the maximum likelihood. Strains are designated by accession number, country/district, and strain name. The 24 strains analyzed in this study are indicated by (♦). The two major groups were identified as A and B.</p

Primers used for RT–qPCR.

<p>Primers used for RT–qPCR.</p

Index of amino acid substitution propensity.

<p>(A) Analysis of 34 PCMV strains from China. (B) Analysis of global PCMV strains.</p

GO annotation of host target genes of differentially expressed miRNAs.

<p>GO functional analysis using the DAVID web-based tool shows that a series of targets belong to a series of functional genes involved in cellular process, metabolic process, and immune system process, which indicated the potential regulatory role of the differentially expressed miRNAs in viral infection. For other enriched GO terms, please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120377#pone.0120377.s006" target="_blank">S4 Table</a>.</p