Validation of predicted <i>ssc-miR-24</i> target genes.

<p>To confirm the legitimacy of selected miR-24 binding sites, a previously developed [REF] in vitro luciferase assay was utilized. Gateway cloning (Life Technologies) was used to create RCAS viruses expressing either the <i>ssc-miR-24</i> hairpin or a scrambled control (<i>SC</i>) hairpin. DF1 cells, a chicken embryonic fibroblast cell line, were infected with either the RCAS-<i>miR-24</i> expressing virus or the RCAS-<i>SC</i> expressing virus were transfected with predicted target 3′ UTR luciferase constructs. Target constructs were generated by cloning the miRNA binding site and its flanking regions downstream of the Renilla luciferase gene in the psiCHECK2 vector (Promega). Relative <i>Renilla</i> luciferase activity is shown. *: p<0.05, **: p<0.01. ^aThis construct contains two <i>miR-24</i> binding sites. ^bThis construct contains two <i>miR-24</i> binding sites. ^cThis construct contains three <i>miR-24</i> binding sites.</p

Validation of predicted <i>ssc-miR-147</i> target genes.

<p>An RCAS-based miRNA expression was utilized to authenticate selected predicted <i>miR-147</i> target sites. The hairpin for <i>ssc-miR-147</i> was cloned into RCASBP(A)-YDV using Gateway cloning (Life Technologies). DF1cells ectopically expressing <i>miR-147</i> or a scrambled control (SC) were transfected with psiCHECK2 constructs containing <i>miR-147</i> binding sites cloned downstream of the <i>Renilla</i> luciferase gene. Relative <i>Renilla</i> luciferase activity is shown. *: p<0.05, **: p<0.01. ^aThis construct contains one <i>miR-147</i> binding site. ^bThis construct contains two <i>miR-147</i> binding sites.</p

Computationally predicted porcine <i>miR-146a</i> target sites selected for validation.

a<p>Capitalized text indicates basing pairings with the microRNA.</p

PRRSV <i>N</i> levels in PAMs transfected with either a <i>miR-147</i> mimic or a negative control mimic.

<p>PAMs were transfected with 100<i>miR-147</i> mimic (Dharmacon) or a negative control mimic (Dharmacon) using amine-based transfection. Sixteen hours post-transfection PAMs were infected with PRRSV (VR-2332) at M.O.I. 1. Total RNA was isolated at 8 hpi, 12 hpi and 24 hpi. Real-time RT-PCR analysis of the PRRSV nucleocapsid protein mRNA levels in PAMs transfected with either a <i>miR-147</i> mimic (grey bars) or a negative control mimic (black bars). *p≤0.05, **p≤0.01.</p

Most frequently sequenced miRNAs in swine alveolar macrophages^a.

a<p>Small RNA libraries were generated from alveolar macrophages from three 7-week old pigs and cultured for 24 hours.</p>b<p>Values are given as average counts per million (cpm) reads.</p>c<p>percentage of total normalized cpm reads matching known and homologous miRNAs.</p

Differentially expressed cellular miRNAs in PRRSV-infected PAMs.

<p>PAMs from three 7-week old pigs were infected with PRRSV (strain VR-2332) at M.O.I. 10 and small RNA expression was determined at 12 hpi, 24 hpi, and 48 hpi using Illumina deep sequencing. A total of forty cellular miRNA were significantly differentially expressed within the first 48 hpi (p<0.05) and are listed along the x-axis. The log fold expression differences of these miRNAs are shown on the y-axis. At 12 hpi (blue bars) 19 miRNAs were differentially expressed, at 24 hpi (red bars) 15 miRNAs were differentially expressed and at 48 hpi (green bars) 12 miRNAs were differentially expressed. Among these six miRNA, <i>miR-30a-3p</i>, <i>miR-132</i>, <i>miR-27b*</i>, <i>miR-29b</i>, <i>miR-146a</i> and <i>miR-9-2</i>, were altered at more than one time point.</p

Computationally predicted porcine <i>miR-24</i> target sites chosen for <i>in vitro</i> validation.

a<p>Capitalized text indicates basing pairings with the microRNA.</p

Characterization of the microRNAome in Porcine Reproductive and Respiratory Syndrome Virus Infected Macrophages

<div><p>Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), a member of the arterivirus family, is the causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS). PRRS is characterized by late term abortions and respiratory disease, particularly in young pigs. Small regulatory RNAs termed microRNA (miRNA) are associated with gene regulation at the post-transcriptional level. MiRNAs are known to play many diverse and complex roles in viral infections. To discover the impact of PRRSV infections on the cellular miRNAome, Illumina deep sequencing was used to construct small RNA expression profiles from <i>in vitro</i> cultured PRRSV-infected porcine alveolar macrophages (PAMs). A total of forty cellular miRNAs were significantly differentially expressed within the first 48 hours post infection (hpi). The expression of six miRNAs, <i>miR-30a-3p</i>, <i>miR-132</i>, <i>miR-27b*</i>, <i>miR-29b</i>, <i>miR-146a</i> and <i>miR-9-2</i>, were altered at more than one time point. Target gene identification suggests that these miRNAs are involved in regulating immune signaling pathways, cytokine, and transcription factor production. The most highly repressed miRNA at 24 hpi was <i>miR-147</i>. A <i>miR-147</i> mimic was utilized to maintain <i>miR-147</i> levels in PRRSV-infected PAMs. PRRSV replication was negatively impacted by high levels of <i>miR-147</i>. Whether down-regulation of <i>miR-147</i> is directly induced by PRRSV or if it is part of the cellular response and PRRSV indirectly benefits remains to be determined. No evidence could be found of PRRSV-encoded miRNAs. Overall, the present study has revealed that a large and diverse group of miRNAs are expressed in swine alveolar macrophages and that the expression of a subset of these miRNAs is altered in PRRSV infected macrophages.</p></div

Over expression of <i>miR-147</i> in PRRSV infected PAMs using a synthetic mimic.

<p>PAMs were transfected with 100<i>miR-147</i> mimic (Dharmacon) or a negative control mimic (Dharmacon) using amine-based transfection. Sixteen hours post-transfection, PAMs were infected with PRRSV (VR-2332) at M.O.I. 1. A. Real-time RT-PCR analysis of <i>miR-147</i> expression in PAMs transfected with either the <i>miR-147</i> mimic (grey bars) or the negative control mimic (black bars). ***p≤0.001. B. Real-time RT-PCR analysis of <i>Snapin</i>, a <i>miR-147</i> target, in PRRSV-infected PAMs transfected with a <i>miR-147</i> mimic (grey bars) or a negative control mimic (black bars). *p≤0.05, **p≤0.01.</p

PRRSV titers in PAMs transfected with either a miR-147 mimic or a negative control mimic.

<p>PAMs were transfected with 100-147 mimic (Dharmacon) or a negative control mimic (Dharmacon). PAMs were infected with PRRSV (VR-2332) at M.O.I. 1. Supernatants were collected at 8 hpi, 12 hpi, and 24 hpi. TCID₅₀ viral titers were determined using the Reed- Muench method and immunofluorescence staining of the PRRSV nucleocapsid protein. Assay was repeated to confirm results.</p