Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3+CD8high cells starting from 14 dpi. Although CD3+CD8high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3+CD8high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages

Brain age as a surrogate marker for cognitive performance in multiple sclerosis

Background: Data from neuro-imaging techniques allow us to estimate a brain's age. Brain age is easily interpretable as "how old the brain looks", and could therefore be an attractive communication tool for brain health in clinical practice. This study aimed to investigate its clinical utility by investigating the relationship between brain age and cognitive performance in multiple sclerosis (MS). Methods: A linear regression model was trained to predict age from brain MRI volumetric features and sex in a healthy control dataset (HC_train, n=1673). This model was used to predict brain age in two test sets: HC_test (n=50) and MS_test (n=201). Brain-Predicted Age Difference (BPAD) was calculated as BPAD=brain age minus chronological age. Cognitive performance was assessed by the Symbol Digit Modalities Test (SDMT). Results: Brain age was significantly related to SDMT scores in the MS_test dataset (r=-0.46, p<.001), and contributed uniquely to variance in SDMT beyond chronological age, reflected by a significant correlation between BPAD and SDMT (r=-0.24, p<.001) and a significant weight (-0.25, p=0.002) in a multivariate regression equation with age. Conclusions: Brain age is a candidate biomarker for cognitive dysfunction in MS and an easy to grasp metric for brain health

Genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is a major cause of economic loss for the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) triggers weak and atypical innate immune responses, but key genes and mechanisms by which the virus interferes with the host innate immunity have not yet been elucidated. In this study, genes that control the response of the main target of PRRSV, porcine alveolar macrophages (PAMs), were profiled in vitro with a time-course experiment spanning the first round of virus replication. PAMs were obtained from six piglets and challenged with the Lelystad PRRSV strain, and gene expression was investigated using Affymetrix microarrays and real-time PCR. Of the 1409 differentially expressed transcripts identified by analysis of variance, two, five, 25, 16 and 100 differed from controls by a minimum of 1.5-fold at 1, 3, 6, 9 and 12 h post-infection (p.i.), respectively. A PRRSV infection effect was detectable between 3 and 6 h p.i., and was characterized by a consistent downregulation of gene expression, followed by the start of the host innate immune response at 9 h p.i. The expression of beta interferon 1 (IFN-β), but not of IFN-α, was strongly upregulated, whilst few genes commonly expressed in response to viral infections and/or induced by interferons were found to be differentially expressed. A predominance of anti-apoptotic transcripts (e.g. interleukin-10), a shift towards a T-helper cell type 2 response and a weak upregulation of tumour necrosis factor-α expression were observed within 12 h p.i., reinforcing the hypotheses that PRRSV has developed sophisticated mechanisms to escape the host defence

Understanding PRRSV Infection in Porcine Lung Based on Genome-Wide Transcriptome Response Identified by Deep Sequencing

Porcine reproductive and respiratory syndrome (PRRS) has been one of the most economically important diseases affecting swine industry worldwide and causes great economic losses each year. PRRS virus (PRRSV) replicates mainly in porcine alveolar macrophages (PAMs) and dendritic cells (DCs) and develops persistent infections, antibody-dependent enhancement (ADE), interstitial pneumonia and immunosuppression. But the molecular mechanisms of PRRSV infection still are poorly understood. Here we report on the first genome-wide host transcriptional responses to classical North American type PRRSV (N-PRRSV) strain CH 1a infection using Solexa/Illumina's digital gene expression (DGE) system, a tag-based high-throughput transcriptome sequencing method, and analyse systematically the relationship between pulmonary gene expression profiles after N-PRRSV infection and infection pathology. Our results suggest that N-PRRSV appeared to utilize multiple strategies for its replication and spread in infected pigs, including subverting host innate immune response, inducing an anti-apoptotic and anti-inflammatory state as well as developing ADE. Upregulation expression of virus-induced pro-inflammatory cytokines, chemokines, adhesion molecules and inflammatory enzymes and inflammatory cells, antibodies, complement activation were likely to result in the development of inflammatory responses during N-PRRSV infection processes. N-PRRSV-induced immunosuppression might be mediated by apoptosis of infected cells, which caused depletion of immune cells and induced an anti-inflammatory cytokine response in which they were unable to eradicate the primary infection. Our systems analysis will benefit for better understanding the molecular pathogenesis of N-PRRSV infection, developing novel antiviral therapies and identifying genetic components for swine resistance/susceptibility to PRRS

The M/GP5 Glycoprotein Complex of Porcine Reproductive and Respiratory Syndrome Virus Binds the Sialoadhesin Receptor in a Sialic Acid-Dependent Manner

The porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to swine health worldwide and is considered the most significant viral disease in the swine industry today. In past years, studies on the entry of the virus into its host cell have led to the identification of a number of essential virus receptors and entry mediators. However, viral counterparts for these molecules have remained elusive and this has made rational development of new generation vaccines impossible. The main objective of this study was to identify the viral counterparts for sialoadhesin, a crucial PRRSV receptor on macrophages. For this purpose, a soluble form of sialoadhesin was constructed and validated. The soluble sialoadhesin could bind PRRSV in a sialic acid-dependent manner and could neutralize PRRSV infection of macrophages, thereby confirming the role of sialoadhesin as an essential PRRSV receptor on macrophages. Although sialic acids are present on the GP3, GP4 and GP5 envelope glycoproteins, only the M/GP5 glycoprotein complex of PRRSV was identified as a ligand for sialoadhesin. The interaction was found to be dependent on the sialic acid binding capacity of sialoadhesin and on the presence of sialic acids on GP5. These findings not only contribute to a better understanding of PRRSV biology, but the knowledge and tools generated in this study also hold the key to the development of a new generation of PRRSV vaccines

Combining Laboratory and Mathematical Models to Infer Mechanisms Underlying Kinetic Changes in Macrophage Susceptibility to an RNA Virus

Background: Macrophages are essential to innate immunity against many pathogens, but some pathogens also target macrophages as routes to infection. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is an RNA virus that infects porcine alveolar macrophages (PAMs) causing devastating impact on global pig production. Identifying the cellular mechanisms that mediate PAM susceptibility to the virus is crucial for developing effective interventions. Previous evidence suggests that the scavenger receptor CD163 is essential for productive infection of PAMs with PRRSV. Here we use an integrative in-vitro-in-silico modelling approach to determine whether and how PAM susceptibility to PRRSV changes over time, to assess the role of CD163 expression on such changes, and to infer other potential causative mechanisms altering cell susceptibility. Results: Our in-vitro experiment showed that PAM susceptibility to PRRSV changed considerably over incubation time. Moreover, an increasing proportion of PAMs apparently lacking CD163 were found susceptible to PRRSV at the later incubation stages, thus conflicting with current understanding that CD163 is essential for productive infection of PAMs with PRRSV. We developed process based dynamic mathematical models and fitted these to the data to assess alternative hypotheses regarding potential underlying mechanisms for the observed susceptibility and biomarker trends. The models informed by our data support the hypothesis that although CD163 may have enhanced cell susceptibility, it was not essential for productive infection in our study. Instead the models promote the existence of a reversible cellular state, such as macrophage polarization, mediated in a density dependent manner by autocrine factors, to be responsible for the observed kinetics in cell susceptibility. Conclusions: Our dynamic model-inference approach provides strong support that PAM susceptibility to the PRRS virus is transient, reversible and can be mediated by compounds produced by the target cells themselves, and that these can render PAMs lacking the CD163 receptor susceptible to PRRSV. The results have implications for the development of therapeutics aiming to boost target cell resistance and prompt future investigation of dynamic changes in macrophage susceptibility to PRRSV and other viruses