Transcription analysis on response of swine lung to H1N1 swine influenza virus

Abstract

Background: As a mild, highly contagious, respiratory disease, swine influenza always damages the innate immune systems, and increases susceptibility to secondary infections which results in considerable morbidity and mortality in pigs. Nevertheless, the systematical host response of pigs to swine influenza virus infection remains largely unknown. To explore it, a time-course gene expression profiling was performed for comprehensive analysis of the global host response induced by H1N1 swine influenza virus in pigs.

Results: At the early stage of H1N1 swine virus infection, pigs were suffering mild respiratory symptoms and pathological changes. A total of 268 porcine genes showing differential expression (DE) after inoculation were identified to compare with the controls on day 3 post infection (PID) (Fold change ≥ 2, p < 0.05). The DE genes were involved in many vital functional classes, mainly including signal transduction, immune response, inflammatory response, cell adhesion and cell-cell signalling. Noticeably, the genes associated with immune and inflammatory response showed highly overexpressed. Through the pathway analysis, the significant pathways mainly concerned with Cell adhesion molecules, Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway and MAPK signaling pathway, suggesting that the host took different strategies to activate these pathways so as to prevent virus infections at the early stage. However, on PID 7, the predominant function classes of DE genes included signal transduction, metabolism, transcription, development and transport. Furthermore, the most significant pathways switched to PPAR signaling pathway and complement and coagulation cascades, showing that the host might start to repair excessive tissue damage by anti-inflammatory functions. These results on PID 7 demonstrated beneficial turnover for host to prevent excessive inflammatory damage and recover the normal state by activating these clusters of genes.

Conclusions: This study shows how the target organ responds to H1N1 swine influenza virus infection in pigs. The observed gene expression profile could help to screen the potential host agents for reducing the prevalence of swine influenza virus and further understand the molecular pathogenesis associated with H1N1 infection in pigs.

Figure 1

The macroscopic and microscopic lung lesions in pigs infected with H1N1 SwIV. (A) Pigs were PBS-mock infected or infected with H1N1 SwIV. On PID 3 and 7, extensive areas of macroscopic lesions of lungs tissues were observed in lungs of virus-infected pigs. (B) Hematoxylin- and eosin-stained sections of lungs from control pigs and the pigs infected with H1N1 SwIV. The microscopic lesions of lung tissues collected on PID 3 and 7 showed acute pneumonia with pathological changes: alveolar wall thickening, bleeding, debris in the lumen, and the accumulation of inflammatory cells, indicated by an arrow (panel ii and iii). The lungs of PBS-mock infected pigs showed normal bronchial epithelial lining and absence of inflammatory cells (panel i).

Figure 2

Clustering and characterization of the differential expression of genes. (A) The DE genes showing clear functional annotation on PID 3 have been selected for cluster analysis which is described in methods. Amongst the DE genes on PID 3, a set of 160 genes were up-regulated and the rest of 108 genes were down-regulated. Each row represents a separate gene and each column represents a separate piglet. Color legend is on the left, the color scale ranges from saturated green for log ratios -3.0 and above to saturated red for log ratios 3.0 and above. Red indicates increased gene expression levels; green indicates decreased levels compared with normal samples. (B) Categories of annotated genes based on biological process GO term. On PID 3, the DE genes mainly clustered into 34 functional groups with varied numbers. (C) The genes showing clear functional annotation on PID 7 have been selected for cluster analysis. (D) On PID 7, the DE genes mainly clustered into 33 functional groups with varied numbers. Many categories shared the same genes.

Figure 3

Validation of the microarray data by qRT-PCR analyses. The genes of TAP1, SAA2, C4, CD2, IRG6, TCN1, ISG20, PR39, PMAP23 and NPG4 were up regulated in SwIV infected pigs compared with normal controls; TGFB2, VEGFA, TLR5 were down regulated. Fold change is calculated based on the mean intensity value from 3 pigs by using the comparative Ct method. Significant levels were analyzed by T-test.

Figure 4

The validation of Toll-like receptors and IFN-stimulated genes by qRT-PCR analyses. (A) The genes in this bioset [differentially regulated by 2-fold or more (P < 0.05)] were clustered according to a hierarchical algorithm; red bars represent up-regulated levels of the 15 representative genes after H1N1 infection, including TLRs and interferon-induced genes (ISGs) which were involved in anti-viral signal molecular or effectors. (B) The genes of TLRs and interferon-induced genes (ISGs) were up-regulated at various degrees in H1N1 SwIV infected pigs compared with the normal controls.