Type I and III IFNs Produced by Plasmacytoid Dendritic Cells in Response to a Member of the Flaviviridae Suppress Cellular Immune Responses.

The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for ∼5-7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro. We further show that the majority of the IFN produced in response to infection both in vitro and in vivo is type III IFN and acid labile. Further, we show IL-28B (IFN-λ3) mRNA is induced in this cell population in vitro. Supernatant from plasmacytoid DCs harvested postinfection with BVDV or recombinant bovine IFN-α or human IL-28B significantly reduced CD4(+) T cell proliferation induced by tubercle bacillus Ag 85-stimulated monocyte-derived DCs. Furthermore, these IFNs induced IFN-stimulated gene expression predominantly in monocyte-derived DCs. IFN-treated immature DCs derived from murine bone marrow also had a reduced capacity to stimulate T cell proliferative responses to tubercle bacillus Ag 85. Immature DCs derived from either source had a reduced capacity for Ag uptake following IFN treatment that is dose dependent. Immunosuppression is a feature of a number of pestivirus infections; our studies suggest type III IFN production plays a key role in the pathogenesis of this family of viruses. Overall, in a natural host, we have demonstrated a link between the induction of type I and III IFN after acute viral infection and transient immunosuppression.

Mechanism of collapse of endoplasmic reticulum cisternae during African swine fever virus infection.

Infection of cells with African swine fever virus (ASFV) can lead to the formation of zipper-like stacks of structural proteins attached to collapsed endoplasmic reticulum (ER) cisternae. We show that the collapse of ER cisternae observed during ASFV infection is dependent on the viral envelope protein, J13Lp. Expression of J13Lp alone in cells is sufficient to induce collapsed ER cisternae. Collapse was dependent on a cysteine residue in the N-terminal domain of J13Lp exposed to the ER lumen. Luminal collapse was also dependent on the expression of J13Lp within stacks of ER where antiparallel interactions between the cytoplasmic domains of J13Lp orientated N-terminal domains across ER cisternae. Cisternal collapse was then driven by disulphide bonds between N-terminal domains arranged in antiparallel arrays across the ER lumen. This provides a novel mechanism for biogenesis of modified stacks of ER present in cells infected with ASFV, and may also be relevant to cellular processes.

Laboratory animal models to study foot-and-mouth disease: a review with emphasis on natural and vaccine-induced immunity.

Laboratory animal models have provided valuable insight into foot-and-mouth disease virus (FMDV) pathogenesis in epidemiologically important target species. While not perfect, these models have delivered an accelerated time frame to characterize the immune responses in natural hosts and a platform to evaluate therapeutics and vaccine candidates at a reduced cost. Further expansion of these models in mice has allowed access to genetic mutations not available for target species, providing a powerful and versatile experimental system to interrogate the immune response to FMDV and to target more expensive studies in natural hosts. The purpose of this review is to describe commonly used FMDV infection models in laboratory animals and to cite examples of when these models have failed or successfully provided insight relevant for target species, with an emphasis on natural and vaccine-induced immunity.

CD4+ T-cell responses to foot-and-mouth disease virus in vaccinated cattle.

We have performed a series of studies to investigate the role of CD4(+) T-cells in the immune response to foot-and-mouth disease virus (FMDV) post-vaccination. Virus neutralizing antibody titres (VNT) in cattle vaccinated with killed FMD commercial vaccine were significantly reduced and class switching delayed as a consequence of rigorous in vivo CD4(+) T-cell depletion. Further studies were performed to examine whether the magnitude of T-cell proliferative responses correlated with the antibody responses. FMD vaccination was found to induce T-cell proliferative responses, with CD4(+) T-cells responding specifically to the FMDV antigen. In addition, gamma interferon (IFN-γ) was detected in the supernatant of FMDV antigen-stimulated PBMC and purified CD4(+) T-cells from vaccinated cattle. Similarly, intracellular IFN-γ could be detected specifically in purified CD4(+) T-cells after restimulation. It was not possible to correlate in vitro proliferative responses or IFN-γ production of PBMC with VNT, probably as a consequence of the induction of T-independent and T-dependent antibody responses and antigen non-specific T-cell responses. However, our studies demonstrate the importance of stimulating CD4(+) T-cell responses for the induction of optimum antibody responses to FMD-killed vaccines.

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness.

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3-4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models.

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection.

Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in naïve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.

Foot-and-mouth disease virus can induce a specific and rapid CD4+ T-cell-independent neutralizing and isotype class-switched antibody response in naïve cattle.

The role of T-lymphocyte subsets in recovery from foot-and-mouth disease virus (FMDV) infection in calves was investigated by administering subset-specific monoclonal antibodies. The depletion of circulating CD4(+) or WC1(+) gammadelta T cells was achieved for a period extending from before challenge to after resolution of viremia and peak clinical signs, whereas CD8(+) cell depletion was only partial. The depletion of CD4(+) cells was also confirmed by analysis of lymph node biopsy specimens 5 days postchallenge. Depletion with anti-WC1 and anti-CD8 antibodies had no effect on the kinetics of infection, clinical signs, and immune responses following FMDV infection. Three of the four CD4(+) T-cell-depleted calves failed to generate an antibody response to the nonstructural polyprotein 3ABC but generated a neutralizing antibody response similar to that in the controls, including rapid isotype switching to immunoglobulin G antibody. We conclude that antibody responses to sites on the surface of the virus capsid are T cell independent, whereas those directed against the nonstructural proteins are T cell dependent. CD4 depletion was found to substantially inhibit antibody responses to the G-H peptide loop VP1(135-156) on the viral capsid, indicating that responses to this particular site, which has a more mobile structure than other neutralizing sites on the virus capsid, are T cell dependent. The depletion of CD4(+) T cells had no adverse effect on the magnitude or duration of clinical signs or clearance of virus from the circulation. Overall, we conclude that CD4(+) T-cell-independent antibody responses play a major role in the resolution of foot-and-mouth disease in cattle.

Foot-and-mouth disease virus persists in the light zone of germinal centres.

Foot-and-mouth disease virus (FMDV) is one of the most contagious viruses of animals and is recognised as the most important constraint to international trade in animals and animal products. Two fundamental problems remain to be understood before more effective control measures can be put in place. These problems are the FMDV "carrier state" and the short duration of immunity after vaccination which contrasts with prolonged immunity after natural infection. Here we show by laser capture microdissection in combination with quantitative real-time reverse transcription polymerase chain reaction, immunohistochemical analysis and corroborate by in situ hybridization that FMDV locates rapidly to, and is maintained in, the light zone of germinal centres following primary infection of naïve cattle. We propose that maintenance of non-replicating FMDV in these sites represents a source of persisting infectious virus and also contributes to the generation of long-lasting antibody responses against neutralising epitopes of the virus.

Reduced redox potential of the cytosol is important for African swine fever virus capsid assembly and maturation.

Assembly of African swine fever virus (ASFV) involves the transfer of the major capsid protein, p73, from the cytosol onto the cytoplasmic face of endoplasmic reticulum-derived membranes. During this process, the folding of p73 is dependent upon transient association with a specific viral chaperone, CAP80. The cell cytoplasm maintains high concentrations of reduced glutathione, leading to a reducing environment. Here, the effects of redox environment on the assembly of ASFV have been studied. Diamide, which oxidizes the cell cytosol, slowed the folding of p73 and prevented release from CAP80 and subsequent binding of p73 to membranes. Similarly, cell oxidation slowed the assembly of p73 molecules already bound to membranes into virus capsid precursors. Interestingly, addition of oxidized glutathione to newly assembled virus capsid precursors in vitro led to disassembly; however, virus particles released from cells were resistant to oxidized glutathione. These data show that assembly of ASFV requires the reducing environment that prevails in the cytosol, but as the virus matures, it becomes resistant to oxidation, possibly indicating preparation for release from the cell.

African swine fever virus induces filopodia-like projections at the plasma membrane.

When exiting the cell vaccinia virus induces actin polymerization and formation of a characteristic actin tail on the cytosolic face of the plasma membrane, directly beneath the extracellular particle. The actin tail acts to propel the virus away from the cell surface to enhance its cell-to-cell spread. We now demonstrate that African swine fever virus (ASFV), a member of the Asfarviridae family, also stimulates the polymerization of actin at the cell surface. Intracellular ASFV particles project out at the tip of long filopodia-like protrusions, at an average rate of 1.8 microm min(-1). Actin was arranged in long unbranched parallel arrays inside these virus-tipped projections. In contrast to vaccinia, this outward movement did not involve recruitment of Grb2, Nck1 or N-WASP. Actin polymerization was not nucleated by virus particles in transit to the cell periphery, and projections were not produced when the secretory pathway was disrupted by brefeldin A treatment. Our results show that when ASFV particles reach the plasma membrane they induce a localized nucleation of actin, and that this process requires interaction with virus-encoded and/or host proteins at the plasma membrane. We suggest that ASFV represents a valuable new model for studying pathways that regulate the formation of filopodia.

Vimentin rearrangement during African swine fever virus infection involves retrograde transport along microtubules and phosphorylation of vimentin by calcium calmodulin kinase II.

African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an "aster" within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly.

Membrane association facilitates the correct processing of pp220 during production of the major matrix proteins of African swine fever virus.

The African swine fever (ASF) virus polyprotein pp220 is processed at Gly-Gly-X sites by a virally encoded SUMO-like protease to produce matrix proteins p150, p37, p34, and p14. Four Gly-Gly-X sites are used to produce the matrix proteins, but the polyprotein contains an additional 15 sites potentially recognized by the protease. This study shows that cleavage occurs at many, if not all, Gly-Gly-X sites, and at steady state, p150 and p34 are minor products of processing. Significantly, only the final structural proteins, p150 and p34, were found in mature virions, suggesting that there is a mechanism for excluding incorrectly processed forms. ASF virus is assembled on the cytoplasmic face of the endoplasmic reticulum, and the distribution of pp220 products between membranes and cytosol was studied. Incorrectly processed forms of p34 were recovered from both the cytosol and membrane fractions. Interestingly, p34 was only detected in the membrane fraction, and of the many processed forms bound to membranes, only p34 was protected from trypsin, suggesting envelopment. The majority of the incorrectly processed forms of p150 were recovered from the cytosol. Again, the correct product of processing, p150, was selectively recruited to membranes. Sucrose density centrifugation showed that membrane-associated forms of p34 and p150 assembled into large structures suggestive of a viral matrix, while cytosolic and/or incorrectly processed forms of pp220 did not. Taken together, these results suggest that association with cellular membranes is important for regulating the correct processing of pp220 and the packaging of matrix proteins into virions.