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Classical Swine Fever Virus Npro Limits Type I Interferon Induction in Plasmacytoid Dendritic Cells by Interacting with Interferon Regulatory Factor 7

ABSTRACT Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus Npro antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here, we report that Npro of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate that the Zn-binding domain of Npro is essential for the interaction of Npro with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required for the interaction with Npro. Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with lower levels of IRF7 in pDC. Consequently, wild-type but not Npro mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV Npro is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the innate defense.

Vector-free transmission and persistence of Japanese encephalitis virus in pigs

Japanese encephalitis virus (JEV), a main cause of severe viral encephalitis in humans, has a complex ecology, composed of a cycle involving primarily waterbirds and mosquitoes, as well as a cycle involving pigs as amplifying hosts. To date, JEV transmission has been exclusively described as being mosquito-mediated. Here we demonstrate that JEV can be transmitted between pigs in the absence of arthropod vectors. Pigs shed virus in oronasal secretions and are highly susceptible to oronasal infection. Clinical symptoms, virus tropism and central nervous system histological lesions are similar in pigs infected through needle, contact or oronasal inoculation. In all cases, a particularly important site of replication are the tonsils, in which JEV is found to persist for at least 25 days despite the presence of high levels of neutralizing antibodies. Our findings could have a major impact on the ecology of JEV in temperate regions with short mosquito seasons.

Efficient Sensing of Infected Cells in Absence of Virus Particles by Blasmacytoid Dendritic Cells Is Blocked by the Viral Ribonuclease Erns

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells, using a virion-free pathway. Here, we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles or infectious RNA which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells was mediated by a contact-dependent RNA transfer to pDC, which was sensitive to a TLR7 inhibitor. This was inhibited by drugs affecting the cytoskeleton and membrane cholesterol. We further demonstrate that a unique viral protein with ribonuclease activity, the viral Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. We propose that this pathway of activation could be of particular importance for viruses which tend to be mostly cell-associated, cause persistent infection, and are non-cytopathogenic.

High interferon type I responses in the lung, plasma and spleen during highly pathogenic H5N1 infection of chicken.

This study shows that high pathogenic H5N1 influenza virus infection of chicken induced high levels of bioactive interferon type I in the lung (4.3 × 105 U/mg tissue), plasma (1.1 × 105 U/mL), and spleen (9.1 × 105 U/mg tissue). In contrast, a low pathogenic attenuated H5N1 vaccine strain only induced approximately 24 times less IFN in the lung, 441 times less in the spleen and 649 less in the plasma. This was in the same range as a reassortant carrying the HA from the vaccine strain and the remaining genes from the high pathogenic virus. On the other hand, a reassortant virus with the HA from the high pathogenic H5N1 with the remaining genes from the vaccine strain had intermediate levels of IFN. The level of interferon responses related to the viral load, and those in the spleen and blood to the spread of virus to lymphoid tissue, as well as disease severity. In vitro, the viruses did not induce interferon in chicken embryonic fibroblasts, but high levels in splenocytes, with not clear relationship to pathogenicity and virulence. This, and the responses also with inactivated viruses imply the presence of plasmacytoid dendritic cell-like leukocytes within the chicken immune system, possibly responsible for the high interferon responses during H5N1 infection. Our data also indicate that the viral load as well as the cleavability of the HA enabling systemic spread of the virus are two major factors controlling systemic IFN responses in chicken.

Interplay of foot-and-mouth disease virus, antibodies and plasmacytoid dendritic cells: virus opsonization under non-neutralizing conditions results in enhanced interferon-alpha responses

Foot-and-mouth disease virus (FMDV) is a highly infectious member of the Picornaviridae inducing an acute disease of cloven-hoofed species. Vaccine-induced immune protection correlates with the presence of high levels of neutralizing antibodies but also opsonising antibodies have been proposed as an important mechanism of the immune response contributing to virus clearance by macrophages and leading to the production of type-I interferon (IFN) by plasmacytoid dendritic cells (pDC). The present study demonstrates that the opsonising antibody titres mediating enhanced IFN-α responses in pDC were similar to neutralizing titres, when antigenically related viruses from the same serotype were employed. However, sera cross-reacted also with non-neutralized isolates of multiple serotypes, when tested in this assay. Both uncomplexed virus and immune complexed virus stimulated pDC via Toll-like receptor 7. An additional finding of potential importance for strain-specific differences in virulence and/or immunogenicity was that pDC activation by FMDV strongly differed between viral isolates. Altogether, our results indicate that opsonising antibodies can have a broader reactivity than neutralizing antibodies and may contribute to antiviral responses induced against antigenically distant viruses.

Characterization and Transcriptomic Analysis of Porcine Blood Conventional and Plasmacytoid Dendritic Cells Reveals Striking Species-Specific Differences.

Porcine dendritic cells (DCs) are relatively well characterized, but a clear-cut identification of all DC subsets combined with full transcriptional profiling was lacking, preventing an unbiased insight into the functional specializations of DC subsets. Using a large panel of Abs in multicolor flow cytometry, cell sorting, and RNA sequencing we identified and characterized the porcine equivalent of conventional DCs (cDC) 1 and cDC2 as well as plasmacytoid DCs (pDCs) in the peripheral blood of pigs. We demonstrate that cDC1 are CD135+CD14−CD172alowCADM1+wCD11R1+ cells, cDC2 are CD135+CD14−CD172a+CADM1+CD115+wCD11R1+CD1+ cells and pDCs are CD4+CD135+CD172a+CD123+CD303+ cells. As described in other species, only cDC1 express BATF3 and XCR1, cDC2 express FCER1A and FCGR2B, and only pDCs express TCF4 and NRP1. Nevertheless, despite these cross-species conserved subset-specific transcripts, porcine pDCs differed from the species described so far in many expressed genes and transcriptomic profiling clustered pDCs more distantly from cDCs than monocytes. The response of porcine DC subsets to TLR ligands revealed that pDCs are by far the most important source of TNF-α, IL-12p40, and of course IFN-α, whereas cDCs are most efficient in MHC and costimulatory molecule expression. Nevertheless, upregulation of CD40 and CD86 in cDCs was critically influenced or even dependent on the presence of pDCs, particularly for TLR 7 and 9 ligands. Our data demonstrate that extrapolation of data on DC biology from one species to another has to be done with care, and it shows how functional details have evolved differentially in different species.

Porcine Cathelicidins Efficiently Complex and Deliver Nucleic Acids to Plasmacytoid Dendritic Cells and Can Thereby Mediate Bacteria-Induced IFN-α Responses

Cathelicidins constitute potent antimicrobial peptides characterized by a high cationic charge that enables strong interactions with nucleic acids. In fact, the only human cathelicidin LL-37 triggers rapid sensing of nucleic acids by plasmacytoid dendritic cells (pDC). Among the porcine cathelicidins, phylogenetic analysis of the C-terminal mature peptide showed that porcine myeloid antimicrobial peptide (PMAP)-36 was the most closely related of the 11 porcine cathelicidins to human LL-37. Despite several investigations evaluating potent antimicrobial functions of porcine cathelicidins, nothing is known about their ability to promote pDC activation. We therefore investigated the capacity of the proline-arginine–rich 39-aa peptide, PMAP-23, PMAP-36, and protegrin-1 to complex with bacterial DNA or synthetic RNA molecules and facilitate pDC activation. We demonstrate that these peptides mediate a rapid and efficient uptake of nucleic acids within minutes, followed by robust IFN-α responses. The highest positively charged cathelicidin, PMAP-36, was found to be the most potent peptide tested for this effect. The peptide–DNA complexes were internalized and also found to associate with the cell membranes of pDC. The amphipathic conformation typical of PMAP-36 was not required for IFN-α induction in pDC. We also demonstrate that PMAP-36 can mediate IFN-α induction in pDC stimulated by Escherichia coli, which alone fail to activate pDC. This response was weaker with a scrambled PMAP-36, relating to its lower antimicrobial activity. Collectively, our data suggest that the antimicrobial and nucleic acid–complexing properties of cathelicidins can mediate pDC activation-promoting adaptive immune responses against microbial infections.

Avian influenza A virus PB2 promotes interferon type I inducing properties of a swine strain in porcine dendritic cells

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.

Porcine B Cell Subset Responses to Toll-like Receptor Ligands

Toll-like receptors (TLR) triggering of B cells are known to promote B cell expansion, differentiation of B cells into antibody-producing and memory cells, but the TLR responses of porcine B cells is poorly characterized. Therefore, this study investigated the response pattern of porcine B cell subsets to a large collection of TLR ligands and demonstrates that the TLR2 ligand Pam3Cys-SK4 and the TLR7/8 ligands gardiquimod and resiquimod are particularly efficient at inducing proliferation, CD25 and CCR7. This activation was also determined in B-cell subpopulations including a CD21+IgM+ subset, an IgG+ subset and two putative B1-like subsets, defined as CD21−IgMhighCD11R1+CD11c+CD14+ and CD21−IgMhigh CD11R1−CD11c+CD14− B cells. The latter two were larger and expressed higher levels of CD80/86 and spontaneous phospholipase C-γ2 phosphorylation. All porcine B-cell subsets were activated by TLR2, TLR7, and TLR9 ligands. Naïve and memory conventional B cells responded similar to TLR ligands. The CD11R1+ B1-like subset had the highest proliferative responses. While both B1-like subsets did not spontaneously secrete IgM, they were the only subsets to produce high level of TLR-induced IgM. Similar to polyclonal IgM responses, memory B cells were efficiently induced to produce specific antibodies by CpG oligodinucleotide, resiquimod, and to a weaker extend by Pam3Cys-SK4. Depletion of plasmacytoid dendritic cells (pDCs) enhanced TLR-induced antibodies. The same set of TLR ligands also induced CD40 on cDCs, pDCs, and monocytes with the exception of TLR4 ligand being unable to activate pDCs. Gardiquimod and resiquimod were particularly efficient at inducing CCR7 on pDCs. Porcine B cells expressed high levels of TLR7, but relatively little other TLR mRNA. Nevertheless, TLR2 on B cells was rapidly upregulated following stimulation, explaining the strong responses following stimulation. Subset-specific analysis of TLR expression demonstrated a comparable expression of TLR2, TLR7, and TLR9 in all B cell subsets, but TLR3 was restricted to B1-like cells, whereas TLR4 was only expressed on conventional B cells, although both at low levels. Altogether, our data describe porcine innate B1-like cells, and how different B cell subsets are involved in innate sensing.

Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies

This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.