RNA-Seq Analysis Reveals Genes Underlying Different Disease Responses to Porcine Circovirus Type 2 in Pigs

Abstract

Porcine circovirus type 2 (PCV2), an economically important pathogen, causes postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as porcine circovirus-associated disease (PCVAD). Previous studies revealed breed-dependent differences in porcine susceptibility to PCV2; however, the genetic mechanism underlying different resistance to PCV2 infection remains largely unknown. In this study, we found that Yorkshire × Landrace (YL) pigs exhibited serious clinical features typifying PCV2 disease, while the Laiwu (a Chinese indigenous pig breed, LW) pigs showed little clinical symptoms of the disease during PCV2 infection. At 35 days post infection (dpi), the PCV2 DNA copy in YL pigs was significantly higher than that in LW pigs (P < 0.05). The serum level of IL-4, IL-6, IL-8, IL-12 and TGF-β1 in LW pigs and TNF-α in YL pigs increased significantly at the early infected stages, respectively; while that of IL-10 and IFN-γ in YL pigs was greatly increased at 35 dpi. RNA-seq analysis revealed that, at 35 dpi, 83 genes were up-regulated and 86 genes were down-regulated in the lung tissues of LW pigs, while in YL pigs, the numbers were 187 and 18, respectively. In LW pigs, the differentially expressed genes (DEGs) were mainly involved in complement and coagulation cascades, metabolism of xenobiotics by cytochrome P450, RIG-I-like receptor signaling and B cell receptor signaling pathways. Four up-regulated genes (TFPI, SERPNC1, SERPNA1, and SERPNA5) that are enriched in complement and coagulation cascades pathway were identified in the PCV2-infected LW pigs, among which the mRNA expression of SERPNA1, as well as three genes including TGF-β1, TGF-β2 and VEGF that are regulated by SERPNA1 was significantly increased (P < 0.05). We speculate that higher expression of SERPNA1 may effectively suppress excessive inflammation reaction and reduce the pathological degree of lung tissue in PCV2-infected pigs. Collectively, our findings indicate that the susceptibility to PCV2 infection depends on a genetic difference between LW and YL pigs, and SERPNA1 likely plays an important role in the resistance of LW pigs to PCV2 infection.

Fig 1. Changes in rectal temperature after PCV2 infection.

The rectal temperature of LW-u pigs (A), LW-i pigs (A), YL-u pigs (B) and YL-i pigs (B) were collected at different dpi. The rectal temperature of YL-i pigs exceeded 40°C from 10 to 21dpi. * P < 0.05 versus YL-u group at same dpi, # P < 0.05 versus YL-i group at 7dpi.

Fig 2. Changes in average daily gain after PCV2 infection.

The average daily gain of LW-u pigs (A), LW-i pigs (A), YL-u pigs (B) and YL-i pigs (B) were collected at different dpi. The average daily gain of YL-i pigs decreased from 14 dpi compared with YL-u pigs. * P < 0.05 versus YL-i group at the same dpi.

Fig 3. Pathological changes in lung tissue after PCV2 infection.

The necropsy (A) and tissue slice (C) results of LW-i pigs showed normal signs. The necropsy (B) and tissue slice (D) results of YL-i pigs showed serious clinical signs: congestion, bleeding, interstitial pneumonia and lymphocyte infiltration in the bronchus determined by hematoxylin and eosin staining (× 200).

Fig 4. Changes in genomic copies of PCV2 in serum after PCV2 infection.

The genomic copies of PCV2 in serum were determined through absolute quantitation PCR. Data were presented as the log10 transformed group means. The PCV2 copies of YL-i pigs were significantly higher than that of LW-i pigs at 35 dpi. * P < 0.05.

Fig 5. Changes in cytokine levels after PCV2 infection.

The fold changes of cytokine levels in serum after PCV2 infection were calculated between LW-i and YL-i pigs. The levels of IL-4 (A), IL-6 (B), IL-8 (C), TGF-β1 (D) and IL-12 (E) in LW pigs were significantly increased at the early stage of PCV2 infection. The level of TNF-α (F) in YL pigs was significantly increased at 4dpi. And the levels of IL-10 (G) and IFN-γ (H) in YL pigs were significantly increased at 35 dpi. * P < 0.05 versus YL (or LW) group at same dpi, # P < 0.05 versus 0 dpi.

Fig 6

Comparison of gene expression levels in the lung tissues between the LW-i and LW-u (A) and YL-i and YL-u (B) libraries. The X and Y-axes show the mRNA expression levels in the two samples. Up-regulated and down-regulated genes were shown in red and green, respectively. Blue dots represent genes with similar expression levels.

Fig 7. Changes in expression of the DEGs mRNA after PCV2 infection.

The mRNA expression of the four DEGs was detected through qPCR. The results of qPCR were similar to the results of RNA-seq. The GAPDH gene was used as reference gene. * P < 0.05.

Fig 8. Changes in SERPINA1 protein expression after PCV2 infection.

Western blotting was used to measure SERPINA1 protein expression in each group of lung tissue samples. The SERPINA1 level in LW-i was significantly higher than that in YL-i. GAPDH expression was used as the positive control. * P < 0.05.

Fig 9. Changes in mRNA expression of SERPINA1-regulated genes after PCV2 infection.

Real-time quantitative RT-PCR was used to assess the mRNA expression levels of TGF-β1/2, VEGF, IL-10, IL-1Ra, TNF-α, TLR2, TLR4 and IL-6 in the lung tissues of the four groups. Increased expression of TGF-β1, TGF-β2 and VEGF mRNA was seen in LW pigs after PCV2 infection. TNF-α, and TLR2 mRNA expression in YL-i was higher than that in LW-i. There was no significant difference in IL-10, IL-1Ra, TLR4 and IL-6 mRNA expression. * P < 0.05.