The results from the present study are in agreement with previous research that demonstrated that H-PRRSV infected pigs exhibit severe clinical symptoms including persistent high fever, reddening of the skin, conjunctivitis, dyspnoea and severe diffuse pulmonary consolidation lesions [2, 22]. Histopathological examination demonstrated robust interstitial pneumonia in the lungs with thickening of alveolar septa accompanied by extensive infiltration of immune cells (Figure 1). The H-PRRSV virus replicates prolifically in the lungs, spleen and lymphoid organs. During infection an invading virus is recognized by PRRs that engage PAMPs and trigger signaling pathways within infected cells that are involved in innate immune (Figure 5) and adaptive immune (Figure 6) responses. Host immune responses are normally protective but if numerous cells are infected before immune induction, immune-mediated destruction can result in severe or fatal pathological consequences. Global profiling of transcriptional changes occurring in host lungs during H-PRRSV viral infection, analyzed using high-throughput Solexa sequencing, has provided important information regarding how H-PRRSV viruses trigger and regulate host immune responses and cause disease.
QPCR assays demonstrated that the H-PRRSV virus replicated rapidly and persisted in infected cells (Table S1 in Additional file 1). Substantial viral antigen was detected in alveolar cells and bronchiolar epithelial cells (Figure 1). The ability of a virus to induce and sustain an infection depends partly on its ability to block host innate immune responses or to modulate the activity of antiviral effector proteins. Production of type I IFN (IFN-α/β, SPI IFN) is an innate antiviral immune reaction in virus-infected cells that prevents viral replication and restricts the spread of the virus to neighboring cells. However, the present study demonstrated that H-PRRSV infection suppressed production of SPI IFN and down-regulated expression of IFN-α (Figure 5E). Previous in vitro and in vivo studies [8, 23, 24] have demonstrated that PRRSV infection results in minimal IFN-α production or suppresses its production, and IFN-α has been shown to inhibit PRRSV replication. During H-RRRSV viral infection, blocking SPI IFN production and particularly production of IFN-α could result in rapid spread of the virus and a high rate of viral replication. Other viral infections including the 1918 influenza virus , hepatitis C virus (HCV)  and Ebola virus  suppress type I IFN gene expression, leading to extensive viral replication and increased pathogenesis. IRF3 plays an important role in typeI IFN gene expression and the present study demonstrated that IRF3 gene expression was suppressed during H-PRRSV infection (Figure 5D). This result is in agreement with a previous study reporting that PRRSV NSP1β inhibited IRF3 and NF-κB transactivation, and down-regulated IFN-β gene expression. This suggested that NSP1β mediates subversion of the host innate immune response and plays an important role in PRRSV pathogenesis. Furthermore, influenza A NSP1 can suppress innate immunity by inhibiting activation of IRF3, and subsequently disrupting the induction of α/β-interferon .
Many viruses induce apoptosis in infected cells but some can block the apoptosis pathway, leading to prolonged life of the cell and an increase in the yield of progeny virions. H-PRRSV up-regulated expression of anti-apoptotic genes and down-regulated expression of some pro-apoptotic genes in H-PRRSV infected lungs (Figure 7C). MCL1, BFL-1, putative inhibitor of apoptosis, ADM and IL10 were up-regulated. MCL1 and BFL-1 belong to the BCL-2 subfamily, which negatively regulates apoptosis and blocks the apoptosis pathway; ADM is an anti-apoptotic peptide ; and IL10 protects cells against apoptosis . The pro-apoptotic genes APR-1, p53 protein, SARP-3, and NDK-H 5 were down-regulated to prevent the occurrence of apoptosis. These findings indicate that H-PRRSV could induce an anti-apoptotic state to prolong the life-span of infected cells and increase the yield of progeny virions.
IL10 could have an important role in the regulation of the immune response to PRRSV. Up-regulation of IL10 gene expression has been demonstrated in PRRSV-infected porcine leukocytes, alveolar macrophages, dendritic cells, and in vivo in PRRSV infected pigs [8, 31]. Incubation of freshly isolated CD14 positive cells with IL10 during differentiation increased susceptibility to PRRSV infection and was correlated with up-regulation of CD163 on the cell surface . This suggests that IL10 plays an important role in CD163 up-regulation and susceptibility to PRRSV during differentiation of macrophages in vivo. CD163 alone can confer PRRSV replication on a non-permissive pig cell line and its expression on macrophages in vivo could determine the efficiency of replication and subsequent pathogenicity of PRRSV . It is possible that internalization of H-PRRSV via CD163 on the target cells could induce expression of IL10 and subsequently induce the expression of CD163 on neighboring undifferentiated monocytes, increasing overall susceptibility to PRRSV.
Taken together, the above findings suggested that the H-PRRSV virus aggressively replicated and disseminated by subverting the host innate immune response, inducing an anti-apoptotic state and up-regulating expression of CD163.
Prolific replication and rapid spread of H-PRRSV virus caused severe lung damage, hemorrhage and extensive infiltration of immune cells throughout the course of infection. Accordingly, significant increases in the expression of a number of genes involved in phagocytic cell activation were observed including CAMs, and several pro-inflammatory cytokines and chemokines such as IFN-γ, TNF, SELL, ICAM, integrin, C-type lectin, IL2RG, IL8, CSF2, IRG6, macrophage inflammatory protein 3 (MIP-3), CXCL2, CXCL9, CXCL10, CCL2 and CCR5 (Figure 5H, J, K). Up-regulated expression of these genes resulted in recruitment of neutrophils, macrophages and other immune cells to sites of infection, and excessive infiltration resulted in destruction of tissues . Moreover, H-PRRSV infection resulted in the activation of CD4 and CD8 T lymphocytes specific for H-PRRSV antigens, and these secreted vasoactive cytokines including TNFα and IFN-γ. This cytokine 'storm' increased capillary fragility (with associated hemorrhages) and permeability. H-PRRSV infection activated complement proteins, which enhanced vascular permeability and were associated with sequestration of thrombocytes. The sustained induction of pro-inflammatory cytokines and chemokines contributed to a robust inflammatory response in the lung.
Fever is frequently the initial response to infection and it is triggered by PRR-PAMP interactions that activate a signaling cascade that causes the production of inflammatory cytokines responsible for fever including CASP1, the IL1-converting enzyme responsible for cleaving the IL-1β precursor and resulting in production of the mature form . TLR2, 4, 6, 7, 9 (Figure 5A) and CASP1 (Figure 7E) were significantly up-regulated in H-PRRSV infected lungs. Heat shock proteins, referred to as stress proteins, are induced in cells exposed to a wide range of environmental stressors including infection and extreme temperature. Gene expression levels of heat shock genes including HSPA5, HSP27, HSP90, HSP90B1, HSPCB and HSPD1 were significantly elevated in H-PRRSV infected lungs relative to C (Figure S7 in Additional file 1).
During H-RRRSV virus infection, activated CTLs and NK cells release perforin and granzymes to kill target cells. Gene expression of PRF1 and granzyme B, A and H were significantly up-regulated in H-PRRSV infected lungs (Figure 6H). Perforin is exocytosed and polymerizes in the target cell plasma membrane to form pores. Granzymes enter target cells through the perforin pores and induce target cell apoptosis. The perforin pores also allow the release of intracellular calcium from the target cell, which acts to trigger apoptotic pathways. The induction of a CTL response results in the release of various cytokines from Th cells, some of which result in clonal proliferation of antigen-specific CTLs, and others that have direct antiviral effects. Diffusion of perforin and local cytokine production frequently results in inflammation and bystander cell damage.
There was up-regulation of genes involved in TNF signaling including TNFα and TNFRSF1A (Figure 7A) and the up-regulation of TNF during PRRSV infection has been reported to have an important role in pathogenicity. It has been reported that PRRSV infection is a potent inducer of TNFα in PAMs . In the present study, continuously up-regulated expression of TNFα (at mRNA and protein levels) from 96 h pi to 168 h pi was observed (Figure 4B). Interestingly, infection with H-PRRSV led to up-regulation of NFKBIA, an inhibitor of the TNF receptor activated transcription factor NF-κB. Loss of NF-κB activity has been reported to increase the cytotoxic effects of TNF and result in increased cell death . TNF and NFKBIA could act synergistically to cause significant alveolar and bronchial epithelial cell necrosis during H-PRRSV infections.
This study has indicated that H-PRRSV could induce apoptosis through a mitochondria-mediated pathway, and previous research provided evidence that PRRSV induces apoptosis in MARC-145 cells through an intrinsic mitochondria-mediated pathway . Pro-apoptotic genes (BAX, BAK, BID, PIK3C3), cytochrome C, and caspases (CASP-10, CASP1, CASP4, CASP15, CASP3) were up-regulated (Figure 7). These results indicate that up-regulation of pro-apoptotic genes resulted in disruption of the mitochondrial transmembrane potential, thereby inducing release of cytochrome c, AIF-like mitochondrion-associated inducer of death and CASP3 from mitochondrial membranes, leading to induction of apoptosis and secondary necrosis. The release of cytochrome c can also induce necrosis through a slower non-apoptotic mechanism due to the electrochemical gradient across the inner membrane, production of reactive oxygen species (ROS) and declining ATP production . The production of ROS, particularly superoxide radicals, and the subsequent oxidative damage to cells and tissues, are recognized as key contributors to viral pathogenesis [36, 39]. ROS-mediated oxidative stress could also contribute to PRRSV-induced apoptosis . In the current study, continuous up-regulated expression of cytochrome b245 heavy chain (GP91-PHOX), a critical component of the membrane-bound oxidase of phagocytes (macrophages and neutrophils) that generates superoxide radicals, was observed from 96 h pi to 168 h pi (Figure 7F). Increased expression of cytochrome b245 in H-PRRSV infected lungs implies the increased production of oxygen radicals and the activation of phagocytic cells. Taken together, these data suggested that the severe pulmonary pathology caused by H-PRRSV infection was induced by significant production of TNF, PRF1, granzymes, cytochrome c and oxygen radicals.