Interferon-α-modulatory sequences from the genome of porcine circovirus type 2

Porcine circovirus (PCV) type 2 is an emerging pathogen among pigs, which has been associated with several severe disease syndromes. To date little is known of the pathogenesis and epidemiology of PCV, and how the virus affects the immune system of the host. To evaluate one possible mechanism of pathogenesis, the genome of PCV-2 was examined for content of CpG-motifs. Five 20 nucleotide long sequences from the genome were tested for their ability to induce production of IFN-a by porcine peripheral blood mononuclear cells (PBMC). One of the oligodeoxynucleotides (ODNs) proved to inhibit the IFN-a production induced by the other ODNs that were stimulatory. The inhibitory ODN (PCV-2/1) was tested against other known inducers of IFN-a and showed a variable degree of inhibitory action depending on the construct of the inducer. ODNs containing phosphorothioate backbone and poly- G- sequences seemed more resistant to inhibition. Also, the inhibitory activity of ODN PCV-2/1 differed against the viral inducers Aujeszky´s disease virus (ADV) and Sendai virus (SV), and the plasmid pcDNA3. Inhibition was most effective against ADV, moderately effective against pcDNA3, but did not affect the IFN-a production induced by SV. The variation in sensitivity to inhibition among the agents could be due to differences in target cell populations. The presence of immune modulatory sequences in the genome of PCV-2 could possibly explain parts of the pathogenesis of the virus.Porcint circovirus (PCV) typ 2 har på senare tid uppmärksammats i växande omfattning, och har associerats med flera allvarliga sjukdomssyndrom hos gris. Man vet fortfarande mycket lite om virusets patogenes och epidemiologi, och hur det påverkar immunsystemet i värddjuret. För att undersöka en möjlig mekanism i patogenesen studerades genomet av PCV-2 med avseende på förekomst av CpGmotiv. Fem sekvenser om 20 nukleotider vardera valdes ut från genomet och deras förmåga att inducera IFN-a i perifera mononukleära blodceller (PBMC) testades. En av oligonukleotiderna (oligos) visade sig kunna hämma IFN-a produktionen som inducerats av de övriga fyra oligos. Den hämmande oligon (PCV-2/1) testades mot andra typer av kända inducerare och visade en varierande förmåga att inhibera IFN-a produktion beroende på konstruktionen av induceraren. Oligo vars kedja till en del utgjordes av fosfotioater istället för fosfodiestrar, samt innehöll upprepade G- sekvenser I 3´- änden verkade mer resistenta mot den inhibitoriska aktivitet hos PCV-2/1. Dessutom var PCV-2/1 inhiberande i varierande grad mot olika typer av virala och bakteriella inducerare. Aujeszky´s disease virus (ADV) hämmades effektivt medan plasmiden pcDNA3 bara hämmades delvis. IFN-a produktionen inducerad av Sendaivirus (SV) påverkades inte av PCV-2/1. Mekanismerna bakom denna variation är inte känd, men kan bero på vilka cellpopulationer som aktiveras av induceraren. Fyndet att PCV-2-genomet innehåller IFN-a-modulerande sekvenser kan bidra till ökad förståelse av virusets patogenes

Dynamics of serum antibodies to and load of porcine circovirus type 2 (PCV2) in pigs in three finishing herds, affected or not by postweaning multisystemic wasting syndrome

Background: Despite that PMWS commonly affects pigs aged eight to sixteen weeks; most studies of PMWS have been conducted during the period before transfer to finishing herds. This study focused on PCV2 load and antibody dynamics in finishing herds with different PMWS status. Methods: Sequentially collected blood samples from 40 pigs in each of two Swedish (A and B) and one Norwegian (C) finishing herds were analysed for serum PCV2-load and -antibodies and saliva cortisol. The two Swedish herds differed in PMWS status, despite receiving animals from the same sow pool (multi-site production). However, the PMWS-deemed herd (A) had previously also received pigs from the spot market. ResultsThe initial serum PCV2 load was similar in the two Swedish herds. In herd A, it peaked after two weeks in the finishing herd and a high number of the pigs had serum PCV2 levels above 10(7) per ml. The antibody titres increased continually with exception for the pigs that developed PMWS, that had initially low and then declining antibody levels. Pigs in the healthy herd B also expressed high titres of antibodies to PCV2 on arrival but remained at that level throughout the study whereas the viral load steadily decreased. No PCV2 antibodies and only low amounts of PCV2 DNA were detected in serum collected during the first five weeks in the PMWS-free herd C. Thereafter a peak in serum PCV2 load accompanied by an antibody response was recorded. PCV2 from the two Swedish herds grouped into genotype PCV2b whereas the Norwegian isolate grouped into PCV2a. Cortisol levels were lower in herd C than in herds A and B. Conclusions: The most obvious difference between the Swedish finishing herds and the Norwegian herd was the time of infection with PCV2 in relation to the time of allocation, as well as the genotype of PCV2. Clinical PMWS was preceded by low levels of serum antibodies and a high load of PCV2 but did not develop in all such animals. It is notable that herd A became affected by PMWS after errors in management routine, emphasising the importance of proper hygiene and general disease-preventing measures

Global gene expression profiling of myeloid immune cell subsets in response to in vitro challenge with porcine circovirus 2b

Compelling evidence suggests that the early interaction between porcine circovirus 2 (PCV-2) and the innate immune system is the key event in the pathogenesis of Post-Weaning Multisystemic Wasting Syndrome (PMWS). Furthermore, PCV2 has been detected in bone-marrow samples, potentially enabling an easy spread and reservoir for the virus. To assess the gene-expression differences induced by an in-vitro PCV2b infection in different three different myeloid innate immune cell subsets generated from the same animal, we used the Agilent Porcine Gene Expression Microarray (V2). Alveolar macrophages (AMØs), monocyte-derived dendritic cells (MoDCs) and bone-marrow cells (BMCs) were generated from each animal, and challenged with a UK-isolate of a PCV2 genotype b-strain at a MOI of 0.5. Remarkably, analysis showed a highly distinct and cell-type dependent response to PCV2b challenge. Overall, MoDCs showed the most marked response to PCV2b challenge in vitro and revealed a key role for TNF in the interaction with PCV2b, whereas only few genes were affected in BMCs and AMØs. These observations were further supported by an enrichment of genes in the downstream NF-κB Signalling pathway as well as an up regulation of genes with pro-apoptotic functions post-challenge. PCV2b challenge increases the expression of a large number of immune-related and pro-apoptotic genes mainly in MoDC, which possibly explain the increased inflammation, granulomatous inflammation and lymphocyte depletion seen in PMWS-affected pigs

Interaction between porcine circovirus type 2 and the immune system of the pig With special reference to immunomodulatory sequences in the viral genome

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) in growing pigs but generally requires the presence of other infectious or non-infectious factors to produce the full clinical expression of disease. These factors remain to be identified, but management routines, concurrent infections and maternal immunity are thought to influence the risk for PMWS outbreak in swine herds. Pigs presenting with PMWS develop severe immunosuppression and the work of this thesis focuses on the interaction between PCV2 and the immune system of the pig in vivo and in vitro. Development of PMWS was studied in models for experimental infection in young pigs using coinfections with porcine parvovirus (PPV) or Escherichia coli. Three-day-old colostrum-deprived pigs infected with PCV2 and PPV developed severe PMWS, and a possible role for interleukin 10 as well as impaired production of antibodies to PCV2 in the development of disease was observed. Four-week-old colostrum fed pigs infected with PCV2 in combination with E. coli did not develop clinical disease, but alterations of functions of immune cells were observed ex vivo and could be related to the presence of PCV2. DNA from virus and bacteria can act modulatory on immune cells through the interaction between unmethylated CpG motifs and toll-like receptor (TLR) 9. Five sequences with interferon (IFN) alpha-modulatory activity were identified in the genome of PCV2, and the inhibitory characteristics of one of these (ODN PCV2/1) were studied further in vitro. The ODN was an efficient inhibitor of IFN-α production induced in porcine peripheral blood mononuclear cells (PBMCs) by other DNA ODNs as well as bacterial or viral DNA, but could not influence the IFN-α production induced by synthetic or viral RNA. The inhibition was dependent on the ability of ODN PCV2/1 to form secondary structures, but did not require the presence of an unmethylated CpG motif. ODN PCV2/1 also inhibited the expression of mRNA for other porcine cytokines by PBMC stimulated in vitro. The presence of immunomodulatory sequences in the genome of PCV2 may contribute to evasive mechanisms utilized by the virus during persistent infection of immune cells and/or development of clinical disease

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs

A new emerging genotype subgroup within PCV-2b dominates the PMWS epizooty in Switzerland

Postweaning multisystemic wasting syndrome (PMWS) is among the most important emerging pig diseases worldwide. Initially, the insidious nature of the disease made it difficult to pinpoint the pathogen. The presence of porcine circovirus type 2 (PCV2) in all PMWS diseased animals led to its acceptance, possibly together with an unknown factor, as the causative agent for PMWS. Also, presence of PCV2 in healthy individuals did not facilitate the understanding of the disease. Phylogenetic classification separates PCV2 viruses into at least two major groups. With the aid of a signature motif, a short amino acid motif encoded within the capsid protein, the viruses are determined as belonging to PCV-2a or PCV-2b. Recently, this classification received more attention, as it seemed to define PCV-2b to be more virulent. This simplification, however, could not be confirmed experimentally. Hence, we investigated whether virus genetic shift was an initiator for the PMWS epizooty in Switzerland. Piglet lymphoid tissues from 1973 to 2005 were investigated by histology, immunohistochemistry (IHC) and PCR. For genotype classification, a sequence amplificate of 137bp was used encompassing the signature motif. The onset of Swiss PMWS epizooty exhibited a marked shift in PMWS diseased and subclinically infected piglets to PCV-2b and specifically to one genotype subgroup. Complementary to these observations, healthy piglets also defined by IHC as negative are positive in the PCR reaction and are void of any PCV-2b virus during epizooty. Consequently, our data support PCV2 genome plasticity as a major contributing factor for PMWS disease manifestation

Expression Dynamics of Innate Immunity in Influenza Virus-Infected Swine

The current circulating swine influenza virus (IV) subtypes in Europe (H1N1, H1N2, and H3N2) are associated with clinical outbreaks of disease. However, we showed that pigs could be susceptible to other IV strains that are able to cross the species barrier. In this work, we extended our investigations into whether different IV strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. For this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a H3N2 Swine IV and four different H3N8 IV strains circulating in different animal species. Pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. In the present study, all groups but mock exhibited antibody responses to IV nucleoprotein protein. Pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. Interestingly, pigs infected with avian and seal H3N8 strains also showed moderate lesions and viral replication, whereas equine and canine IVs did not cause overt pathological signs, and replication was barely detectable. Swine IV infection induced interferon (IFN)-alpha and interleukin-6 responses in bronchoalveolar fluids (BALF) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. However, IFN-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of IFN-alpha genes. Remarkably, the Equine strain gave rise to a Serum Amyloid A response in BALF despite little if any replication. Each virus strain could be associated with expression of cytokine genes and/or proteins after infection. These responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system