Understanding PRRSV infection in porcine lung based on genome-wide transcriptome response identified by deep sequencing

Abstract

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS.

Figure 1. Pathologic examination of lungs infected with the N-PRRSV on day 7 post-infection.

(A) Normal morphology observed in UNC porcine lung; (B) Interstitial pneumonia in the lung with thickening of alveolar septa accompanied with infiltration of immune cells; (C) Most viral antigen (brown) was detected in the alveolar cells and the bronchiolar epithelial cells in lesions.

Figure 2. qPCR validation of DGE data.

Relative quantitation was carried out to measure changes in target gene expression in lung samples relative to an endogenous reference sample. Results are expressed as the target/reference ratio of each sample normalized by the target/reference ratio of the calibrator. HPRT1 was used as a reference gene. The vertical axis indicates the fold change of transcript abundance in N-PRRSV infected pigs compared to the UNC. For the C sample, the fold change of transcript abundance relative to the C sample equals one, by definition. qPCR-B: the RNA samples from independent RNA extractions from biological replicates; qPCR-P:the RNA samples from pooling samples that were used for deep sequencing. Error bars represent SE.

Figure 3. Differential expression genes related to the regulation of replication and spread of N-PRRSV, anorexia, slow growth.

(A) Interferons and associated genes; (B) Upregulated genes involved in lipid metabolism; (C) Downregulated genes associated with sterol, cholesterol, and lipid biosynthetic and metabolic process; (D) Genes involved in the induction of anti-apoptotic state; (E) Genes associated with anorexia and (F) subsequent slow growth. Genes shown in red were upregulated and those shown in green were downregulated in infected relative to UNC pigs. See supplementary Table S3 for full gene names.

Figure 4. Differential expression in functional processes related to fever.

(A) Fever-related genes; (B) Heat shock genes. The red and green color represent significantly induced or repressed gene expression, respectively. See supplementary Table S3 for full gene names.

Figure 5. Expression of genes involved in inflammatory response in N-PRRSV-infected porcine lungs.

(A) Cell surface and cytoplasmic pattern-recognition receptors (PRRs); (B) IFN-regulatory factors (IRFs) and signal transducer and activator of transcription (STATs); (C) Interferon-stimulated genes (ISGs); (D) Cytokines; (E) Chemokines; (F) Adhesion molecules and inflammatory enzymes; (G) Immunoglobulin; (H) Fc receptors and mannose receptor C1 (MRC1); (I) Complement. See supplementary Table S3 for full gene names.

Figure 6. Expression of selected cell death and tissues damage -related genes in N-PRRSV-infected porcine lungs.

(A) Ubiquitin-proteins and ubiquitin enzymes; (B) Proteasomes; (C) Genes relative to assembling and transport of MHC-I-peptide complex; (D) Cathepsins; (E) MHC class II antigens; (F) Costimulatory molecules and cell adhesion molecules (CAMs); (G) TCRs/CD3 complex and co-receptor molecules;(H) Perforin (PFR) and granzymes; (I) Pro-apoptotic genes; (J) Oxidative stress-related genes. See supplementary Table S3 for full gene names.

Figure 7. Model of the relationship between pulmonary gene expression profiles and infection pathology.

Genes shown in red were upregulated and those shown in green were downregulated in infected relative to UNC pigs.