Project description:Background: Porcine respiratory and reproductive syndrome virus (PRRSV) is a single stranded RNA virus member that infects pigs and causes losses to the commercial industry reaching upwards of a billion dollars annually in combined direct and indirect costs. The virus can be separated into etiologies that contain multiple heterologous low and highly pathogenic strains. Recently the United States has begun to see an increase in heterologous type 2 PRRSV strains of higher virulence (HP-PRRSV). The high pathogenicity of these strains can drastically alter host immune responses and the ability of the animal to maintain homeostasis. Because the loss of host homeostasis can denote underlying changes in gene and regulatory element expression profiles, the study aimed to examine the effect PRRSV infections has on miRNA and tRNA expression and the roles they play in host tolerance or susceptibility. Results: Using transcriptomic analysis of whole blood taken from control and infected pigs at several timepoints (1, 3, 8 dpi), the analysis returned a total of 149 statistically significant (FDR ⫹ 0.15) miRNAs (n=89) and tRNAs (n=60) that were evaluated for possible pro and anti-viral effects. The tRNA differential expression increased in both magnitude and count as dpi increased, with no statistically significant expression at 1 dpi. The most abundant tRNA amino acid at 3 dpi was alanine , while glycine was the most abundant at 8 dpi. For the miRNAs, focus was put on upregulation that can inhibit gene expression. These results yielded candidates with potential anti and pro-viral actions such as Ssc-miR-125b, which is predicted to limit PRRSV viral levels, and Ssc-miR-145-5p shown to cause alternative macrophage priming. The results also showed that both the tRNA and miRNA displayed expression patterns Conclusions: The results indicated that the HP-PRRSV infection effects host homeostasis at the epigenetic level through changes in miRNA and tRNA expression and their subsequent gene interactions, that target and influence the function of host immune, metabolic, and structural pathways.

Project description:Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets, and high mortality. In this study, we employed Affymetrix microarray chip technology to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited the range of clinical features that typify the disease, while the DPL pigs exhibited only mild signs of the disease. The percentage of CD8+ T cells in the DPL pigs was significantly higher than that in the DLY pigs at 21 days post-infection (dpi) (p< 0.05). Interleukin (IL) 1 beta (IL-1β) and IL-2 levels showed significant differences between the DPL and DLY pigs at 0 and 7 dpi (p< 0.01). For IL-10, the DLY pigs had significantly higher values than the DPL pigs at 0 and 7 dpi (p< 0.01). Significant differences were apparent between the DPL and DLY pigs in terms of their tumor necrosis factor-alpha (TNF-α) and interferon (IFN)-gamma (IFN-γ) levels at 0 and 7 dpi (p< 0.01). Microarray data revealed 16 differentially expressed genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The expression levels of 10 of the 16 genes, namely CCDC84, C6ORF52, THYMOSIN, PRVE, HSPCB, CYP2J2, AMPD3, TOR1AIP2, PTGES3, and ACOX3, were validated by real-time quantitative RT-PCR. This study provides a platform for further investigation of the molecular mechanisms underlying the differential immune responses to PRRSV infection in different breeds or lines of pig. We investigated the response of lung tissues from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs infected with porcine reproductive and respiratory syndrome virus (strain JXA1) by using the Affymetrix Porcine Genome Array.

Project description:Obese and lean pig breeds showed obvious phenotypic variations and physiological differences in skeletal muscle growth. Prenatal muscle development programs postnatal performance. In this study we initially conducted full transcriptional profiling of prenatal skeletal muscle from Tongcheng pigs (obese) and Landrace pigs (lean) at 33, 65 and 90 days post coitus (dpc), using long serial analysis of gene expression (LongSAGE). We subsequently sequenced 317,115 LongSAGE tags and identified 1400 and 1201 differentially expressed transcripts that showed eight expression patterns for Tongcheng and Landrace, respectively. These two breeds had more significant differences in their gene expression profiles at 65 than that at 33 and 90 dpc. We also identified 532, 653 and 459 transcripts that were differentially expressed at 33, 65 and 90 dpc between the two breeds, respectively. The cellular function of the differentially expressed transcripts that matched annotated genes revealed that each stage had a uniquely altered profile between the two breeds in various functional categories including muscle fiber constitute and contraction, apoptosis, protein synthesis, and signalling transduction. Our results suggest that skeletal muscle development potentially has a greater lag in its growth rate at 33-65 dpc in Tongcheng pigs when compared with their Landrace counterparts. Our analyses therefore not only will provide valuable resources in the further identification of candidate genes for meat production traits, but also assist in the elucidation of the development of prenatal skeletal muscle in pigs and other vertebrates. Keywords: comparative transcriptome analysis Three Tongcheng and Landrace sows were mated with the same boar of each corresponding breed, respectively. The sows were then sacrificed at a commercial slaughterhouse and the uteri containing the embryos were collected immediately. The longissimus muscle tissues were then rapidly dissected from each of the embryos at 33, 65 and 90 dpc for both the Tongcheng and Landrace pigs. These samples were snap-frozen in liquid nitrogen and stored at -80°C until further use in the LongSAGE experiments. For the skeletal muscles from the six different samples, that is, Tongcheng (T33, T65 and T90, respectively) and Landrace (L33, L65 and L90, respectively), equal quantities of total RNA from each individual (n=4) were pooled. Six LongSAGE libraries were subsequently generated using I-SAGETM Long kits (Invitrogen, USA) according to the manufacturer’s instructions. Transforming clones were sequenced with the help of an ABI PRIZM 3730 DNA sequencer.

Project description:Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is an infectious disease characterized by severe reproductive deficiency in pregnant sows, respiratory symptoms in piglets, and high mortality. In this study, we employed Affymetrix microarray chip technology to compare the gene expression profiles of lung tissue samples from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs after infection with PRRSV. During infection with PRRSV, the DLY pigs exhibited the range of clinical features that typify the disease, while the DPL pigs exhibited only mild signs of the disease. The percentage of CD8+ T cells in the DPL pigs was significantly higher than that in the DLY pigs at 21 days post-infection (dpi) (p< 0.05). Interleukin (IL) 1 beta (IL-1β) and IL-2 levels showed significant differences between the DPL and DLY pigs at 0 and 7 dpi (p< 0.01). For IL-10, the DLY pigs had significantly higher values than the DPL pigs at 0 and 7 dpi (p< 0.01). Significant differences were apparent between the DPL and DLY pigs in terms of their tumor necrosis factor-alpha (TNF-α) and interferon (IFN)-gamma (IFN-γ) levels at 0 and 7 dpi (p< 0.01). Microarray data revealed 16 differentially expressed genes in the lung tissue samples from the DLY and DPL pigs (q≤5%), of which LOC100516029 and LOC100523005 were up-regulated in the PRRSV-infected DPL pigs, while the other 14 genes were down-regulated in the PRRSV-infected DPL pigs compared with the PRRSV-infected DLY pigs. The expression levels of 10 of the 16 genes, namely CCDC84, C6ORF52, THYMOSIN, PRVE, HSPCB, CYP2J2, AMPD3, TOR1AIP2, PTGES3, and ACOX3, were validated by real-time quantitative RT-PCR. This study provides a platform for further investigation of the molecular mechanisms underlying the differential immune responses to PRRSV infection in different breeds or lines of pig. We investigated the response of lung tissues from Dapulian (DPL) pigs (a Chinese indigenous pig breed) and Duroc×Landrace×Yorkshire (DLY) pigs infected with porcine reproductive and respiratory syndrome virus (strain JXA1) by using the Affymetrix Porcine Genome Array. Sixteen healthy 30-day-old weaned DPL pigs were selected from the Jiaxiang Dapulian farm, Jining City, China, and 15 healthy 30-day-old weaned DLY pigs were obtained from a commercial farm with high standards of animal health. These pigs were free from PRRSV, porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), and classical swine fever virus (CSFV) as determined by ELISA tests for serum antibodies; the absence of PRRSV was also confirmed by real-time quantitative reverse transcription PCR (qRT-PCR). Pigs were randomly assigned into two groups and reared in separate places: the PRRSV-infected group consisted of 11 DPL and 10 DLY pigs, and the control group consisted of five DPL and five DLY pigs. Infections in the pigs proceeded via inoculation with 2 ml of a viral suspension of PRRSV (at a tissue culture infectious dose of 105) by dripping the solution into the nasal cavity of each pig. The control group was treated with an identical volume of PBS by the same method. Rectal temperatures and clinical examinations on the pigs were recorded daily during the experiment. Anticoagulant-treated blood and untreated blood samples were collected separately at 0, 7, 14, and 21 days post-infection (dpi) from the infected and control groups for assaying CD4+, CD8+, cytokine (interleukin (IL) 1 beta (IL-1β), IL-2, IL-10, interferon (IFN)-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and immunoglobulin G (IgG) protein levels. Lung samples for microarray analysis and real-time qRT-PCR analysis were collected from six infected DLY and DPL pigs (three pigs for each breed) immediately post-slaughter at 28 dpi. Total RNA was isolated from lung tissue samples and purified using an RNeasy Mini kit according to the manufacturer’s protocol. RNA was prepared using the GeneChip (AFF-900623) one cycle target for the labeling and control reagents, and the labeled RNA was hybridized in an Affymetrix Hybridization Oven 640 for sequencing.

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Differential microRNA expression and microRNA editing upon HP-PRRSV infection in vivo across different pig breeds

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