GSE19970
Understanding normal PRRSV infection in lung based on genome-wide transcriptome response identified by deep sequencing
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 reported on the first genome-wide host transcriptional responses to normal PRRSV (N-PRRSV) infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyzed systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results indicated that N-PRRSV appeared to utilize multiple strategies for its long surviral in infected pigs, including subverting host innate immune response, hijacking host lipid metabolism, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes production and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processing. 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 or developed secondary 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. Overall design: Nine conventionally-reared, healthy 6-week-old, crossbred weaned pigs (Landrace×Yorkshire) were selected from a high-health commercial farm.All pigs were PRRSV-seronegative determined by ELISA (HerdChek PRRS 2XR; IDEXX Laboratories) and absence of PRRSV tested by RT-PCR. Pigs were randomly assigned to three groups in the experiment and raised in isolation rooms. Six pigs were inoculated with 6 ml viral suspension (4 ml intranasally and 2 ml intramuscularly) of North American type PRRSV (N-PRRSV, isolated from China) at a dose of 106.0 TCID50 ml-1 on day 0. Three negative control pigs were treated similarly with an identical volume of DMEM culture media from uninfected MARC-145 cells 1 day prior to experimental infection, and were immediately necropsied.N-PRRSV-inoculated pigs were clinically examined daily and rectal body temperatures were recorded from days -2 to 7 post infection (pi).Three infected pigs randomly chosen within each group were necropsied at each time point of 96 h pi and 168 h pi. Lung samples were collected from uninfected negative control group (C), three pigs at 96 h pi (N96), three pigs at 168 h pi (N168) and immediately frozen in liquid nitrogen for RNA isolation or fixed in 10% neutralized buffered formalin for histological processing. Total RNA was extracted from frozen lungs using standard protocols (Trizol) and then treated with DNase to remove potential genomic DNA contamination according to the manufactures's protocols. RNA integrity and concentration were evaluated by Agolent 2100 Bioanalyzer.For RNA library construction and deep sequencing, RNA samples were prepared as follows: for each time point of H-PRRSV-inoculated group and UNC group equal quantities of RNA isolated from three individual lungs were pooled. Approximately 6 µg of RNA representing each group were submitted to Solexa (now Illumina Inc.) for sequencing.