The double-stranded RNA bluetongue virus induces type I interferon in plasmacytoid dendritic cells via a MYD88-dependent TLR7/8-independent signaling pathway.

Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/ß) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/ß production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/ß induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/ß in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/ß. BTV replication in pDCs was not mandatory for IFN-α/ß production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/ß required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/ß induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/ß in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.

Increase in chemokines CXCL10 and CCL2 in blood from pigs infected with high compared to low virulence African swine fever virus isolates.

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.

Involvement of the skin during bluetongue virus infection and replication in the ruminant host.

Bluetongue virus (BTV) is a double stranded (ds) RNA virus (genus Orbivirus; family Reoviridae), which is considered capable of infecting all species of domestic and wild ruminants, although clinical signs are seen mostly in sheep. BTV is arthropod-borne ("arbovirus") and able to productively infect and replicate in many different cell types of both insects and mammalian hosts. Although the organ and cellular tropism of BTV in ruminants has been the subject of several studies, many aspects of its pathogenesis are still poorly understood, partly because of inherent problems in distinguishing between "virus replication" and "virus presence".BTV replication and organ tropism were studied in a wide range of infected sheep tissues, by immuno-fluorescence-labeling of non-structural or structural proteins (NS2 or VP7 and core proteins, respectively) using confocal microscopy to distinguish between virus presence and replication. These results are compared to gross and microscopic pathological findings in selected organs from infected sheep. Replication was demonstrated in two major cell types: vascular endothelial cells, and agranular leukocytes which morphologically resemble lymphocytes, monocytes/macrophages and/or dendritic cells. Two organs (the skin and tonsils) were shown to support relatively high levels of BTV replication, although they have not previously been proposed as important replication sites during BTV infection. The high level of BTV replication in the skin is thought to be of major significance for the pathogenesis and transmission of BTV (via biting insects) and a refinement of our current model of BTV pathogenesis is discussed.

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

Inhibition of a large double-stranded DNA virus by MxA protein.

Increasing evidence points to the importance of the interferon (IFN) response in determining the host range and virulence of African swine fever virus (ASFV). Infection with attenuated strains of ASFV leads to the upregulation of genes controlled by IFN pathways, including myxovirus resistance (Mx) genes that are potent effectors of the antiviral state. Mx gene products are known to inhibit the replication of many negative-sense single-stranded RNA viruses, as well as double-stranded RNA viruses, positive-sense single-stranded RNA viruses, and the reverse-transcribing DNA virus hepatitis B virus. Here, we provide data that extend the known range of viruses inhibited by Mx to include the large double-stranded DNA viruses. Stably transfected Vero cells expressing human MxA protein did not support ASFV plaque formation, and virus replication in these cells was reduced 100-fold compared with that in control cells. In contrast, ASFV replication in cells expressing MxB protein or a mutant MxA protein was similar to that in control Vero cells. There was a drastic reduction in ASFV late protein synthesis in MxA-expressing cells, correlating with the results of previous work on the effect of IFN on viral replication. Strikingly, the inhibition of ASFV replication was linked to the recruitment of MxA protein to perinuclear viral assembly sites, where the protein surrounded the virus factories. Interactions between ASFV and MxA were similar to those seen between MxA and different RNA viruses, suggesting a common inhibitory mechanism.

Bluetongue virus targets conventional dendritic cells in skin lymph.

Bluetongue virus (BTV) is the etiological agent of bluetongue, a hemorrhagic disease of ruminants (particularly sheep), which causes important economic losses around the world. BTV is transmitted primarily via the bites of infected midges, which inject the virus into the ruminant's skin during blood feeding. The virus initially replicates in the draining lymph node and then disseminates to secondary organs where it induces edema, hemorrhages, and necrosis. In this study, we show that ovine conventional dendritic cells (cDCs) are the primary targets of BTV that contribute to the primary dissemination of BTV from the skin to draining lymph nodes. Lymph cDCs support BTV RNA and protein synthesis, as well as the production of infectious virus belonging to several different BTV serotypes, regardless of their level of attenuation. Afferent lymph cell subsets, other than cDCs, showed only marginal levels of BTV protein expression. BTV infection provoked a massive recruitment of cDCs to the sheep skin and afferent lymph, providing cellular targets for infection. Although BTV productively infects cDCs, no negative impact on their physiology was detected. Indeed, BTV infection and protein expression in cDCs enhanced their survival rate. Several serotypes of BTV stimulated the surface expression of the CD80 and CD86 costimulatory molecules on cDCs as well as the mRNA synthesis of cytokines involved in inflammation and immunity, i.e., interleukin-12 (IL-12), IL-1beta, and IL-6. BTV-infected cDCs stimulated antigen-specific CD4 and CD8 proliferation as well as gamma interferon production. BTV initially targets cDCs while preserving their functional properties, reflecting the optimal adaptation of the virus to its host cells for its first spread.

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine.

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells.

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8alpha, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3+CD5+CD6+ T-cells (-40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8alphaalpha and CD8alphabeta lymphocytes, but was not expressed in gammadelta T-cells or monocyte/macrophages. The perforin positive CD3- subset was phenotypically homogeneous and defined as CD5-CD6-CD8beta-CD16+CD11b+. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3+) could be divided into at least three subsets. The first subset was CD4-CD5+CD6+CD11b-CD16- most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8beta. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4+CD5+CD6+CD8beta+CD16-CD11b- and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3+CD4-CD5+CD6-CD8beta+/-CD11b+CD16+, and possessed MHC-unrestricted cytotoxicity and LAK activity.

Correlation of cell surface marker expression with African swine fever virus infection.

The expression of surface markers on African swine fever virus (ASFV) infected cells was evaluated to assess their involvement in infection. Previous findings indicated CD163 expression was correlated with ASFV susceptibility. However, in this study the expression of porcine CD163 on cell lines did not increase the infection rate of these cells indicating other factors are likely to be important in determining susceptibility to infection. On adherent porcine bone marrow (pBM) cells the expression of CD45 was strongly correlated with infection. CD163 and CD203a expression correlated at intermediate levels with infection, indicating cells expressing these markers could become infected but were not preferentially infected by the virus. Most of the cells expressing MHCII were infected, indicating that they may be preferentially infected although expression of MHCII was not essential for infection and a large percentage of the infected cells were MHCII negative. CD16 showed a marked decrease in expression following infection and significantly lower levels of infected cells were shown to express CD16. Altogether these results suggest CD163 may be involved in ASFV infection but it may not be essential; the results also highlight the importance of other cell markers which requiring further investigation.

Modulation of chemokine and chemokine receptor expression following infection of porcine macrophages with African swine fever virus.

African swine fever virus (ASFV) is the only member of the Asfarviridae, a large DNA virus family which replicates predominantly in the cytoplasm. Most isolates cause a fatal haemorrhagic disease in domestic pigs, although some low virulence isolates cause little or no mortality. The modulation of chemokine responses following infection of porcine macrophages with low and high virulence isolates was studied to indicate how this may be involved in the induction of pathogenesis and of effective immune responses. Infection with both low and high virulence isolates resulted in down-regulation of mRNA levels for chemokines CCL2, CCL3L, CXCL2 and chemokine receptors CCR1, CCR5, CXCR3, CXCR4 and up-regulation in expression of mRNAs for CCL4, CXCL10 and chemokine receptor CCR7. Levels of CCL4, CXCL8, CXCL10 mRNAs were higher in macrophages infected with low virulence isolate OURT88/3 compared to high virulence isolate Benin 97/1. Levels of CXCL8 and CCL2 protein were significantly reduced in supernatants from macrophages infected with Benin 97/1 isolate compared to OURT88/3 and mock-infected macrophages. There was also a decreased chemotactic response of donor cells exposed to supernatants from Benin 97/1 infected macrophages compared to those from OURT88/3 and mock-infected macrophages. The data show that infection of macrophages with the low virulence strain OURT88/3 induces higher expression of key inflammatory chemokines compared to infection with high virulence strain Benin 97/1. This may be important for the induction of effective protective immunity that has been observed in pigs immunised with the OURT88/3 isolate.

Deletion of virulence associated genes from attenuated African swine fever virus isolate OUR T88/3 decreases its ability to protect against challenge with virulent virus.

African swine fever virus (ASFV) causes an acute haemorrhagic disease of domestic pigs against which there is no effective vaccine. The attenuated ASFV strain OUR T88/3 has been shown previously to protect vaccinated pigs against challenge with some virulent strains including OUR T88/1. Two genes, DP71L and DP96R were deleted from the OUR T88/3 genome to create recombinant virus OUR T88/3ΔDP2. Deletion of these genes from virulent viruses has previously been shown to reduce ASFV virulence in domestic pigs. Groups of 6 pigs were immunised with deletion virus OUR T88/3ΔDP2 or parental virus OUR T88/3 and challenged with virulent OUR T88/1 virus. Four pigs (66%) were protected by inoculation with the deletion virus OUR T88/3ΔDP2 compared to 100% protection with the parental virus OUR T88/3. Thus the deletion of the two genes DP71L and DP96R from OUR T88/3 strain reduced its ability to protect pigs against challenge with virulent virus.

Cellular immunity in ASFV responses.

African swine fever virus (ASFV) infection usually results in an acute haemorrhagic disease with a mortality rate approaching 100% in domestic pigs. However, pigs can survive infection with less-virulent isolates of ASFV and may become chronically infected. Surviving animals are resistant to challenge with homologous or, in some cases, closely related isolates of the virus indicating that pigs can develop protective immunity against ASFV. During asymptomatic, non-virulent ASFV infections natural killer cell activity increases in pigs, suggesting this cell type plays a role in ASFV immunity. Furthermore, depletion of CD8(+) lymphocytes from ASFV immune pigs demolishes protective immunity against related virulent viruses. This suggests that ASFV specific antibody alone is not sufficient for protection against ASFV infection and that there is an important role for the CD8(+) lymphocyte subset in ASFV protective immunity. These results were supported by DNA immunization studies, demonstrating a correlation between the protection afforded against lethal challenge and the detection of a large number of vaccine-induced antigen-specific CD8(+) T-cells. Peripheral blood mononuclear cells (PBMCs) from ASF immune pigs protected from clinical disease show higher proportions of ASFV specific CD4(+)CD8(high+) double positive cytotoxic T cells than PBMCs from ASF immune but clinically diseased pig. The frequency of ASFV specific IFNγ producing T cells induced by immunization correlates to the degree of protection from ASFV challenge, and this may prove to be a useful indicator of any potential cross-protection against heterologous ASFV isolates.

Protection of IFNAR (-/-) mice against bluetongue virus serotype 8, by heterologous (DNA/rMVA) and homologous (rMVA/rMVA) vaccination, expressing outer-capsid protein VP2.

The protective efficacy of recombinant vaccines expressing serotype 8 bluetongue virus (BTV-8) capsid proteins was tested in a mouse model. The recombinant vaccines comprised plasmid DNA or Modified Vaccinia Ankara viruses encoding BTV VP2, VP5 or VP7 proteins. These constructs were administered alone or in combination using either a homologous prime boost vaccination regime (rMVA/rMVA) or a heterologous vaccination regime (DNA/rMVA). The DNA/rMVA or rMVA/rMVA prime-boost were administered at a three week interval and all of the animals that received VP2 generated neutralising antibodies. The vaccinated and non-vaccinated-control mice were subsequently challenged with a lethal dose of BTV-8. Mice vaccinated with VP7 alone were not protected. However, mice vaccinated with DNA/rMVA or rMVA/rMVA expressing VP2, VP5 and VP7 or VP2 alone were all protected.

Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to increased infectivity specifically for Culicoides cells and, in turn, efficiency of transmission to the insect vector.

Protection of European domestic pigs from virulent African isolates of African swine fever virus by experimental immunisation.

African swine fever (ASF) is an acute haemorrhagic disease of domestic pigs for which there is currently no vaccine. We showed that experimental immunisation of pigs with the non-virulent OURT88/3 genotype I isolate from Portugal followed by the closely related virulent OURT88/1 genotype I isolate could confer protection against challenge with virulent isolates from Africa including the genotype I Benin 97/1 isolate and genotype X Uganda 1965 isolate. This immunisation strategy protected most pigs challenged with either Benin or Uganda from both disease and viraemia. Cross-protection was correlated with the ability of different ASFV isolates to stimulate immune lymphocytes from the OURT88/3 and OURT88/1 immunised pigs.

Viraemia and clinical disease in Dorset Poll sheep following vaccination with live attenuated bluetongue virus vaccines serotypes 16 and 4.

The spread of bluetongue virus (BTV) is most successfully controlled by vaccination of susceptible ruminant populations. Currently two different types of BTV vaccines are used for this purpose; inactivated, mostly monovalent vaccine formulations and modified live virus vaccines (MLVs). Clinical signs and viraemia in Dorset Poll sheep vaccinated with BTV-4 and BTV-16 MLVs or inoculated with homogenates of midges (C. sonorensis and C. nubeculosus) previously infected with BTV-4 MLV are presented. All sheep vaccinated with the two MLVs mounted an infectious viraemia lasting for a minimum of 9 up to 23 days post vaccination and developed a range of clinical signs associated with BTV infection. Peak viraemia titres recorded in individual sheep ranged from 3.5 to 6.83 log(10)TCID(50)/ml indicating a high potential for infection of vector insects and onward transmission. The implications of these results are discussed with reference to the current outbreaks of BTV occurring in northern Europe and in relation to the future development of vaccines for this virus.

Dramatic improvement in FMD DNA vaccine efficacy and cross-serotype antibody induction in pigs following a protein boost.

To overcome the low and slow development of humoral antibody often observed with DNA vaccines we applied a prime-boost strategy. When FMD DNA vaccine P1-2A3C3D and pGM-CSF primed pigs were boosted with inactivated foot-and-mouth disease virus (FMDV) antigen and recombinant 3D (without adjuvant) an average 36-fold increase in the FMDV antibody response was observed compared to conventional vaccination, that included a log(10) virus neutralising titre increase. Most remarkably, a significant level of cross-serotype reactivity was observed against A, C and Asia1 in the virus neutralisation and ELISA tests. This prime-boost strategy fully protected pigs from a heterologous challenge.

Antigen delivery systems for veterinary vaccine development. Viral-vector based delivery systems.

The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.

Enhancing immune responses against a plasmid DNA vaccine encoding a FMDV empty capsid from serotype O.

Various parameters have been examined for the improvement of immune responses induced by immunisation with foot-and-mouth disease virus (FMDV) DNA vaccine (pcDNA 3.1/P1-2A3C3D, P1) in swine. Our findings show that increasing the amount of P1 DNA plasmids and pGMCSF adjuvant plasmids induces stronger FMDV specific and neutralising antibody responses, as well as promoting cytokines IL-8 and IFNgamma secretion, in immunised pigs via multiple inoculation sites.

African swine fever virus causes microtubule-dependent dispersal of the trans-golgi network and slows delivery of membrane protein to the plasma membrane.

Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.