Molecular characterization of transcriptome-wide interactions between highly pathogenic porcine reproductive and respiratory syndrome virus and porcine alveolar macrophages in vivo

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) infects mainly the porcine alveolar macrophages (PAMs) and causes porcine reproductive and respiratory syndrome (PRRS). Previous studies have analyzed the global gene expression profiles of lung tissue in vivo and PAMs in vitro following infection with PRRSV, however, transcriptome-wide understanding of the interaction between highly pathogenic PRRSV (HP-PRRSV) and PAMs in vivo has not yet been established. In this study, we employed Affymetrix microarrays to investigate the gene expression patterns of PAMs isolated from Tongcheng piglets (a Chinese indigenous breed) after infection with HP-PRRSV. During the infection, Tongcheng piglets exhibited typical clinical signs, e.g. fever, asthma, coughing, anorexia, lethargy and convulsion, but displayed mild regional lung damage at 5 and 7 dpi. Microarray analysis revealed that HP-PRRSV infection has affected PAMs in expression of the important genes involved in cytoskeleton and exocytosis organization, protein degradation and folding, intracellular calcium and zinc homeostasis. Several potential antiviral strategies might be employed in PAMs, including upregulating IFN-induced genes and increasing intracellular zinc ion concentration. And inhibition of the complement system likely attenuated the lung damage during HP-PRRSV infection. Transcriptomic analysis of PAMs in vivo could lead to a better understanding of the HP-PRRSV-host interaction, and to the identification of novel antiviral therapies and genetic components of swine tolerance/susceptibility to HP-PRRS.

Keywords: HP-PRRSV-host interaction; antiviral strategy; infection; microarray; pulmonary alveolar macrophage.

Figure 1

Laboratory infection of Tongcheng piglets with HP-PRRSV. A: Rectal temperature of the piglets; B: Viral copy numbers per ml serum determined by absolute real-time quantitative PCR; C: Lung morphology of piglets slaughtered at 0, 5 or 7 dpi; D: Gross lung lesion scores of the piglets. The scores of 0 to 100 denote the different severity of gross lung lesion, from intact to totally damaged; E and F: Paraffin sections of the apex of lung at 0 dpi (E) and 5 dpi (F), stained with hematoxylin and eosin. Scale bars indicate 50 μm.

Figure 2

Microarray analysis of PAMs' transcriptional responses to HP-PRRSV infection. A: Hierarchical clustering analysis of gene expression profiles pre- and post-infection. Each column represents one piglet, and each horizontal line refers to a gene. Color legend is on the top-left of the figure. Red indicates genes with a greater expression relative to the geometrical means, green indicates genes with a lower expression relative to the geometrical means; B: Biological process Gene Ontology (GO) analysis of 166 differentially expressed genes. Many categories shared the same transcripts.

Figure 3

Sensing the HP-PRRSV infection by host cell. PRRSV enters early endosomes but does not continue through the endocytic pathway to late endosomes . The ATP6V1B2 gene, which encodes a component of vacuolar ATPase (V-ATPase) that mediates acidification of endosomal organelles , facilitates the uncoating of the virus. Viral nucleic acids could be sensed by Toll-like receptors (TLRs) pathway or RIG-I pathway both of which lead to type-I IFN induction by activating IRF3 and IRF7, and to inflammatory cytokines expression by activating the MAPK signaling pathway. Several IFN-induced genes were upregulated during HP-PRRSV infection, even though no induction of type-I IFN was observed. Red background in the gene box indicates upregulation of the gene expression, green indicates downregulation, and white indicates no change of the gene expression. Fold changes of the differentially expressed genes are 1.94 (ATP6V1B2), 0.31 (SARM1), 4.58 (IRF7), 1.79 (SOCS1), 1.59 (SBNO2), 1.81 (NMI), and 2.48 (STAT1), respectively.

Figure 4

Q-PCR validation of the microarray data. P values (T-test) of the Q-PCR data are 0.018 (CCL2), 0.039 (SLC39A14), 0.044 (DDIT3), 0.006 (GLRX2), 0.008 (ATP6V1B2), 0.508 (TNF) and 0.032 (C3), respectively. TNF is a non-differentially expressed gene.