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Differences in Whole Blood Gene Expression Associated with Infection Time-Course and Extent of Fetal Mortality in a Reproductive Model of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection.

Wilkinson JM, Ladinig A, Bao H, Kommadath A, Stothard P, Lunney JK, Harding JC, Plastow GS - PLoS ONE (2016)

Bottom Line: A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed.Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points.This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada.

ABSTRACT
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant females causes fetal death and increased piglet mortality, but there is substantial variation in the extent of reproductive pathology between individual dams. This study used RNA-sequencing to characterize the whole blood transcriptional response to type 2 PRRSV in pregnant gilts during the first week of infection (at 0, 2, and 6 days post-inoculation), and attempted to identify gene expression signatures associated with a low or high level of fetal mortality rates (LFM and HFM; n = 8/group) at necropsy, 21 days post-inoculation. The initial response to infection measured at 2 days post-inoculation saw an upregulation of genes involved in innate immunity, such as interferon-stimulated antiviral genes and inflammatory markers, and apoptosis. A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed. By day 6 the pattern had reversed, with a drop in innate immune signaling and an increase in the expression of genes involved in cell division and T cell signaling. Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points. Among the 15 DEGs upregulated in LFM gilts on all three days were several genes involved in platelet function, including integrins ITGA2B and ITGB3, and the chemokine PF4 (CXCL4). LFM gilts exhibited a higher baseline expression of interferon-stimulated and pro-inflammatory genes prior to infection, and of T cell markers two days post-infection, indicative of a more rapid progression of the immune response to PRRSV. This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.

No MeSH data available.


Related in: MedlinePlus

Relationship between T Cell and Mitosis Gene Expression Six Days Post-inoculation with PRRSV.(A) T cell receptor signaling pathway from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2 in whole blood of PRRSV-inoculated gilts. Color intensity indicates magnitude of differential expression. (B) Plot of mean T cell (CD3+ve) number for the 16 experimental gilts during the first six days of infection showing the rebound in T cell numbers between day 2 and day 6 post-infection. Cell counts were obtained from a previously published dataset [10]. Asterisks indicate significant cell count differences between successive time-points (P<0.001). (C) Mitosis signaling pathways from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2. Color intensity indicates magnitude of differential expression. The data suggest that mitosis is at least partly responsible for the rebound in T cell numbers between D2 and D6 post-inoculation.
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pone.0153615.g003: Relationship between T Cell and Mitosis Gene Expression Six Days Post-inoculation with PRRSV.(A) T cell receptor signaling pathway from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2 in whole blood of PRRSV-inoculated gilts. Color intensity indicates magnitude of differential expression. (B) Plot of mean T cell (CD3+ve) number for the 16 experimental gilts during the first six days of infection showing the rebound in T cell numbers between day 2 and day 6 post-infection. Cell counts were obtained from a previously published dataset [10]. Asterisks indicate significant cell count differences between successive time-points (P<0.001). (C) Mitosis signaling pathways from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2. Color intensity indicates magnitude of differential expression. The data suggest that mitosis is at least partly responsible for the rebound in T cell numbers between D2 and D6 post-inoculation.

Mentions: At D6, the pattern of gene expression for apoptotic and T cell signaling was reversed from D2: apoptosis signaling was downregulated, while T cell signaling was upregulated. This was accompanied by an increase in T cell numbers, and increase in the expression of genes that feature in pathways associated with mitosis (Fig 3). These included ‘Mitotic roles of polo-like kinase’, ‘Cell cycle G2/M DNA damage checkpoint regulation’, and ‘Cell cycle control of chromosomal replication’. The top gene sets related to genes regulated by two transcription factors involved in cell cycle progression: E2F and MYC. The gene sets ‘DNA Repair’ and ‘Mitotic Spindle’ were also significant. Specific genes included cyclins (CCNB1, CCNB2, CCNB3), cell division regulatory kinases (AURKA, CDC7, CDK1, CHEK1, PKMYT1, PLK1, PLK4, WEE1) and phosphatases (CDC25B, CDC25C), DNA replication complex proteins (CC45, CDC6, MCM2, MCM3, MCM4, ORC6, PCNA, RPA1, RPA3) and chromosome separation proteins (CENPE, CENPM, ESPL1, FBOX5, KIF11, KIF23, PTTG1, TOP2A). This expression profile provided a clear indication of active cellular proliferation, as many of the mechanistic components of mitosis were upregulated. Many of the other most upregulated genes are known to be expressed in T cells such as cytolytic enzymes (GZMA, GZMB, GZMK), and cell surface proteins (CD3D, CD3E, CD3G, CD8B, CTLA4). We previously demonstrated that total leukocyte numbers rebounded in infected gilts at D6 post infection [10], and that the biggest % increase was in the T cell fraction. These expression results support this, and suggest that at least some of this rebound in the blood population is due to active cell proliferation, and not solely to an influx of leukocytes into the bloodstream from other sites. Other studies have also identified an increase in CD8B positive CTL in the peripheral blood of infected pigs at a similar time-point [39–41]. Whether this proliferation is caused by the activation of PRRSV-specific CTL or is a broader response of CTL to an immunostimulatory signal, such as polyclonal activation or cytokine signaling, is not clear. A role for IL10 in this process has previously been postulated [40], but we found that CTL proliferation and IL10 expression were negatively correlated in gilt whole blood. Irrespective of the cause, an expansion in CTL numbers does precede the eventual detection of IFN-γ by PRRSV-specific CTL from 2 weeks post-infection [40]. And although the appearance of this adaptive immune response is delayed and weaker than that of many viral infections, it does coincide with a drop in viremia that could indicate the importance of cell-mediated immunity for virus clearance [40].


Differences in Whole Blood Gene Expression Associated with Infection Time-Course and Extent of Fetal Mortality in a Reproductive Model of Type 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Infection.

Wilkinson JM, Ladinig A, Bao H, Kommadath A, Stothard P, Lunney JK, Harding JC, Plastow GS - PLoS ONE (2016)

Relationship between T Cell and Mitosis Gene Expression Six Days Post-inoculation with PRRSV.(A) T cell receptor signaling pathway from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2 in whole blood of PRRSV-inoculated gilts. Color intensity indicates magnitude of differential expression. (B) Plot of mean T cell (CD3+ve) number for the 16 experimental gilts during the first six days of infection showing the rebound in T cell numbers between day 2 and day 6 post-infection. Cell counts were obtained from a previously published dataset [10]. Asterisks indicate significant cell count differences between successive time-points (P<0.001). (C) Mitosis signaling pathways from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2. Color intensity indicates magnitude of differential expression. The data suggest that mitosis is at least partly responsible for the rebound in T cell numbers between D2 and D6 post-inoculation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836665&req=5

pone.0153615.g003: Relationship between T Cell and Mitosis Gene Expression Six Days Post-inoculation with PRRSV.(A) T cell receptor signaling pathway from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2 in whole blood of PRRSV-inoculated gilts. Color intensity indicates magnitude of differential expression. (B) Plot of mean T cell (CD3+ve) number for the 16 experimental gilts during the first six days of infection showing the rebound in T cell numbers between day 2 and day 6 post-infection. Cell counts were obtained from a previously published dataset [10]. Asterisks indicate significant cell count differences between successive time-points (P<0.001). (C) Mitosis signaling pathways from Ingenuity Pathway Analysis showing genes that are upregulated (red) on D6 compared to D2. Color intensity indicates magnitude of differential expression. The data suggest that mitosis is at least partly responsible for the rebound in T cell numbers between D2 and D6 post-inoculation.
Mentions: At D6, the pattern of gene expression for apoptotic and T cell signaling was reversed from D2: apoptosis signaling was downregulated, while T cell signaling was upregulated. This was accompanied by an increase in T cell numbers, and increase in the expression of genes that feature in pathways associated with mitosis (Fig 3). These included ‘Mitotic roles of polo-like kinase’, ‘Cell cycle G2/M DNA damage checkpoint regulation’, and ‘Cell cycle control of chromosomal replication’. The top gene sets related to genes regulated by two transcription factors involved in cell cycle progression: E2F and MYC. The gene sets ‘DNA Repair’ and ‘Mitotic Spindle’ were also significant. Specific genes included cyclins (CCNB1, CCNB2, CCNB3), cell division regulatory kinases (AURKA, CDC7, CDK1, CHEK1, PKMYT1, PLK1, PLK4, WEE1) and phosphatases (CDC25B, CDC25C), DNA replication complex proteins (CC45, CDC6, MCM2, MCM3, MCM4, ORC6, PCNA, RPA1, RPA3) and chromosome separation proteins (CENPE, CENPM, ESPL1, FBOX5, KIF11, KIF23, PTTG1, TOP2A). This expression profile provided a clear indication of active cellular proliferation, as many of the mechanistic components of mitosis were upregulated. Many of the other most upregulated genes are known to be expressed in T cells such as cytolytic enzymes (GZMA, GZMB, GZMK), and cell surface proteins (CD3D, CD3E, CD3G, CD8B, CTLA4). We previously demonstrated that total leukocyte numbers rebounded in infected gilts at D6 post infection [10], and that the biggest % increase was in the T cell fraction. These expression results support this, and suggest that at least some of this rebound in the blood population is due to active cell proliferation, and not solely to an influx of leukocytes into the bloodstream from other sites. Other studies have also identified an increase in CD8B positive CTL in the peripheral blood of infected pigs at a similar time-point [39–41]. Whether this proliferation is caused by the activation of PRRSV-specific CTL or is a broader response of CTL to an immunostimulatory signal, such as polyclonal activation or cytokine signaling, is not clear. A role for IL10 in this process has previously been postulated [40], but we found that CTL proliferation and IL10 expression were negatively correlated in gilt whole blood. Irrespective of the cause, an expansion in CTL numbers does precede the eventual detection of IFN-γ by PRRSV-specific CTL from 2 weeks post-infection [40]. And although the appearance of this adaptive immune response is delayed and weaker than that of many viral infections, it does coincide with a drop in viremia that could indicate the importance of cell-mediated immunity for virus clearance [40].

Bottom Line: A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed.Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points.This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.

View Article: PubMed Central - PubMed

Affiliation: Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, AB, Canada.

ABSTRACT
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection of pregnant females causes fetal death and increased piglet mortality, but there is substantial variation in the extent of reproductive pathology between individual dams. This study used RNA-sequencing to characterize the whole blood transcriptional response to type 2 PRRSV in pregnant gilts during the first week of infection (at 0, 2, and 6 days post-inoculation), and attempted to identify gene expression signatures associated with a low or high level of fetal mortality rates (LFM and HFM; n = 8/group) at necropsy, 21 days post-inoculation. The initial response to infection measured at 2 days post-inoculation saw an upregulation of genes involved in innate immunity, such as interferon-stimulated antiviral genes and inflammatory markers, and apoptosis. A concomitant decrease in expression of protein synthesis and T lymphocyte markers was observed. By day 6 the pattern had reversed, with a drop in innate immune signaling and an increase in the expression of genes involved in cell division and T cell signaling. Differentially expressed genes (DEGs) associated with extremes of litter mortality rate were identified at all three time-points. Among the 15 DEGs upregulated in LFM gilts on all three days were several genes involved in platelet function, including integrins ITGA2B and ITGB3, and the chemokine PF4 (CXCL4). LFM gilts exhibited a higher baseline expression of interferon-stimulated and pro-inflammatory genes prior to infection, and of T cell markers two days post-infection, indicative of a more rapid progression of the immune response to PRRSV. This study has increased our knowledge of the early response to PRRSV in the blood of pregnant gilts, and could ultimately lead to the development of a biomarker panel that can be used to predict PRRSV-associated reproductive pathology.

No MeSH data available.


Related in: MedlinePlus