Analysis of the genetic diversity and mRNA expression level in porcine reproductive and respiratory syndrome virus vaccinated pigs that developed short or long viremias after challenge.

Porcine reproductive and respiratory syndrome virus (PRRSv) infection alters the host's cellular and humoral immune response. Immunity against PRRSv is multigenic and vary between individuals. The aim of the present study was to compare several genes that encode for molecules involved in the immune response between two groups of vaccinated pigs that experienced short or long viremic periods after PRRSv challenge. These analyses include the sequencing of four SLA Class I, two Class II allele groups, and CD163, plus the analysis by quantitative realtime qRT-PCR of the constitutive expression of TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA and other molecules in peripheral blood mononuclear cells.

Quasispecies evolution of the prototypical genotype 1 porcine reproductive and respiratory syndrome virus early during in vivo infection is rapid and tissue specific.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major infectious threat to the pig industry worldwide. Increasing evidence suggests that microevolution within a quasispecies population can give rise to high sequence heterogeneity in PRRSV; potentially impacting the pathogenicity of the virus. Here, we report on micro-evolutionary events taking place within the viral quasispecies population in lung and lymph node 3 days post infection (dpi) following experimental in vivo infection with the prototypical Lelystad PRRSV (LV). Sequence analysis revealed 16 high frequency single nucleotide variants (SNV) or differences from the reference LV genome which are assumed to be representative of the consensus inoculum genome. Additionally, 49 other low frequency SNVs were also found in the inoculum population. At 3 dpi, a total of 9 and 10 SNVs of varying frequencies could already be detected in the LV population infecting the lung and lymph nodes, respectively. Interestingly, of these, three and four novel SNVs emerged independently in the two respective tissues when compared to the inoculum. The remaining variants, though already present at lower frequencies in the inoculum, were positively selected and their frequency increased within the quasispecies population. Hence, we were able to determine directly from tissues infected with PRRSV the repertoire of genetic variants within the viral quasispecies population. Our data also suggest that microevolution of these variants is rapid and some may be tissue-specific.

Distinct functional enrichment of transcriptional signatures in pigs with high and low IFN-gamma responses after vaccination with a porcine reproductive and respiratory syndrome virus (PRRSV).

Little is known about the host factor in the response to PRRSV vaccination. For this purpose, piglets were immunized with a commercial PRRSV-live vaccine and classified as high responders (HR) or low responders (LR) as regards to the frequencies of virus-specific IFN-γ-secreting cells. Six weeks post vaccination, PBMCs isolated from three individuals with the most extreme responses in each HR and LR groups and 3 unvaccinated controls, were either stimulated with phytohaemagglutinin, challenged with the vaccine or mock treated for 24 h, prior conducting transcriptional studies, gene ontology and pathway analyses. The LR group had very low neutralizing antibody levels and showed a higher number of down-regulated transcripts compared with the HR group (FDR < 0.2, P < 0.001). Down-regulated genes encoded chemoattractants, proinflammatory cytokines and the interferon-inducible GBP family, and showed enrichment in wounding (FDR < 3.6E-13), inflammation (FDR < 8E-12), defence (FDR < 8.7E-09) and immunity (FDR < 7.6E-08), suggesting immune response impairment. In the HR group, down-regulated genes were involved in protein transport (FDR < 4.77E-03), locomotory behavior (FDR < 5.47E-3), regulation of protein localization (FDR < 1.02E-02), and regulation of TNF superfamily member 15 and miR181. In contrast, the HR group presented up-regulated transcripts associated with wounding (FDR < 4.95). Moreover, IFN-γ was predicted to be an inhibited upstream regulator since IFN-γ pathways were associated with higher number of down-regulated genes in the LR (n = 40) than the HR (n = 10). Divergent responses to PRRSV-vaccination may be the result of the genetic background of the host.

Genomic variation in macrophage-cultured European porcine reproductive and respiratory syndrome virus Olot/91 revealed using ultra-deep next generation sequencing.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is a disease of major economic impact worldwide. The etiologic agent of this disease is the PRRS virus (PRRSV). Increasing evidence suggest that microevolution within a coexisting quasispecies population can give rise to high sequence heterogeneity in PRRSV. FINDINGS: We developed a pipeline based on the ultra-deep next generation sequencing approach to first construct the complete genome of a European PRRSV, strain Olot/9, cultured on macrophages and then capture the rare variants representative of the mixed quasispecies population. Olot/91 differs from the reference Lelystad strain by about 5% and a total of 88 variants, with frequencies as low as 1%, were detected in the mixed population. These variants included 16 non-synonymous variants concentrated in the genes encoding structural and nonstructural proteins; including Glycoprotein 2a and 5. CONCLUSION: Using an ultra-deep sequencing methodology, the complete genome of Olot/91 was constructed without any prior knowledge of the sequence. Rare variants that constitute minor fractions of the heterogeneous PRRSV population could successfully be detected to allow further exploration of microevolutionary events.

Down-regulation of mechanisms involved in cell transport and maintenance of mucosal integrity in pigs infected with Lawsonia intracellularis.

Lawsonia intracellularis is an obligate intracellular bacterium, responsible for the disease complex known as proliferative enteropathy (PE). L. intracellularis is associated with intestinal crypt epithelial cell proliferation but the mechanisms responsible are yet to be defined. Microarray analysis was used to investigate the host-pathogen interaction in experimentally infected pigs to identify pathways that may be involved. Ileal samples originating from twenty-eight weaner pigs experimentally challenged with a pure culture of L. intracellularis (strain LR189/5/83) were subjected to microarray analysis. Microarray transcriptional signatures were validated using immunohistochemistry and quantitative real time PCR of selected genes at various time points post challenge. At peak of infection (14 days post challenge) 86% of altered transcripts were down regulated, particularly those involved in maintenance of mucosal integrity and regulation of cell transport. Among the up-regulated transcripts, CD163 and CDK1 were novel findings and considered to be important, due to their respective roles in innate immunity and cellular proliferation. Overall, targeted cellular mechanisms included those that are important in epithelial restitution, migration and protection; maintenance of stable inter-epithelial cell relationships; cell transport of nutrients and electrolytes; innate immunity; and cell cycle.

Host inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Little is known how the virus subverts the innate immune response to initiate its replication in alveolar macrophages. Large-scale transcriptional responses of macrophages with different levels of susceptibility to PRRSV infection were compared over 30 h of infection. This study demonstrates a rapid and intense host transcriptional remodelling during the early phase of the replication of the virus which correlates with transient repression of type-I interferon transcript as early as 8 h post-infection. These results support the suggestion from previous studies that host innate immune response inhibits replication of European porcine reproductive and respiratory syndrome virus in macrophages by altering differential regulation of type-I interferon transcriptional response.

Innate immune responses to replication of porcine reproductive and respiratory syndrome virus in isolated Swine alveolar macrophages.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by a positive RNA strand arterivirus. PRRS virus (PRRSV) interacts primarily with lung macrophages. Identifying the genetic components involved in host resistance/susceptibility would represent an important step forward in the design of disease control programs. In this study, alveolar macrophages derived from five commercial pig lines were used to study the innate immune response to PRRSV infection in vitro. Analysis by flow cytometry has demonstrated that bronchial alveolar lavage fluid (BALF) preparations were almost exclusively composed of alveolar macrophages and that the pigs tested were free from infection. Macrophages from the Landrace line showed significantly reduced virus replication and poor growth of PRRSV during 30 h of infection. By 72 h, PRRSV viral load was down to 2.5 log(10) TCID(50) compared with an average of 5 log(10) TCID(50) for the other breeds tested. These observations suggest that factors intrinsic to the Landrace breed may be responsible for this reduced or delayed response to PRRSV. Preliminary investigation suggests that the PRRSV coreceptor, sialoadhesin, may not be responsible for the Landrace macrophage phenotype as its abundance and localisation were comparable in all the breeds. Strikingly, we found that the reduced or delayed growth of PRRSV was temporally associated with high levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-8 mRNA accumulation and substantial reduction of secretion of IL-8, suggesting a key contributory role for cytokine synthesis and secretion during the innate immune response to PRRSV infection.

Lawsonia intracellularis infection of intestinal crypt cells is associated with specific depletion of secreted MUC2 in goblet cells.

The expression patterns of secreted (MUC2 and MUC5AC) and membrane-tethered (MUC1, MUC4, MUC12 and MUC13) mucins were monitored in healthy pigs and pigs challenged orally with Lawsonia intracellularis. These results showed that the regulation of mucin gene expression is distinctive along the GI tract of the healthy pig, and may reflect an association between the function of the mucin subtypes and different physiological demands at various sites. We identified a specific depletion of secreted MUC2 from goblet cells in infected pigs that correlated with the increased level of intracellular bacteria in crypt cells. We concluded that L. intracellularis may influence MUC2 production, thereby altering the mucus barrier and enabling cellular invasion.

Complete Genome Sequence of a Pathogenic Genotype 1 Subtype 3 Porcine Reproductive and Respiratory Syndrome Virus (Strain SU1-Bel) from Pig Primary Tissue.

We report here the complete genome of the pathogenic eastern European subtype 3 porcine reproductive and respiratory syndrome virus (PRRSV) strain SU1-Bel, sequenced directly from a pig lymph node. While sharing substantial sequence similarity with other subtype 3 strains, SU1-Bel is found to harbor unique indels and contain putative novel subgenomic RNAs.