A replication analysis of foot-and-mouth disease virus in swine lymphoid tissue might indicate a putative carrier stage in pigs

Foot-and-mouth disease virus (FMVD), one of the most contagious viruses of cloven-hoofed animals, may cause a prolonged, asymptomatic but persistent infection in ruminants, named the "carrier state". However, it remains an open question whether this carrier state occurs in pigs. Here we present quantitative analyses of the duration of FMDV RNA and infectivity in lymphoid and epithelial tissues in experimentally infected pigs with FMDV C-S8c1. The data indicated that although FMDV RNA remained in blood until day 14 post-infection (pi), viremia was cleared by day 7 pi. However, all tissues tested were positive for FMDV until day 14-17 pi. Interestingly, the specific infectivity of FMDV in these tissues was in some cases even higher than the FMDV C-S8c1. We therefore propose that a "pseudopersistent state" may occur in pigs in which virus replicates in lymphoid tissues for a prolonged period of time, thereby representing a potential source of virus

Expression of Porcine Fusion Protein IRF7/3(5D) Efficiently Controls Foot-and-Mouth Disease Virus Replication

Several studies have demonstrated that the delivery of type I, II, or III interferons (IFNs) by inoculation of a replication-defective human adenovirus 5 (Ad5) vector expressing IFNs can effectively control foot-and-mouth disease (FMD) in cattle and swine during experimental infections. However, relatively high doses are required to achieve protection. In this study, we identified the functional properties of a porcine fusion protein, poIRF7/3(5D), as a biotherapeutic and enhancer of IFN activity against FMD virus (FMDV). We showed that poIRF7/3(5D) is a potent inducer of type I IFNs, including alpha IFN (IFN-α), IFN-β, and IFN-ω but not type III IFN (interleukin-28B), without inducing cytotoxicity. Expression of poIRF7/3(5D) significantly and steadily reduced FMDV titers by up to 6 log(10) units in swine and bovine cell lines. Treatment with an IFN receptor inhibitor (B18R) combined with an anti-IFN-α antibody neutralized the antiviral activity in the supernatants of cells transduced with an Ad5 vector expressing poIRF7/3(5D) [Ad5-poIRF7/3(5D)]. However, several transcripts with known antiviral function, including type I IFNs, were still highly upregulated (range of increase, 8-fold to over 500-fold) by poIRF7/3(5D) in the presence of B18R. Furthermore, the sera of mice treated with Ad5-poIRF7/3(5D) showed antiviral activity that was associated with the induction of high levels of IFN-α and resulted in complete protection against FMDV challenge at 6, 24, or 48 h posttreatment. This study highlights for the first time the antiviral potential of Ad5-poIRF7/3(5D) in vitro and in vivo against FMDV. IMPORTANCE FMD remains one of the most devastating diseases that affect livestock worldwide. Effective vaccine formulations are available but are serotype specific and require approximately 7 days before they are able to elicit protective immunity. We have shown that vector-delivered IFN is an option to protect animals against many FMDV serotypes as soon as 24 h and for about 4 days postadministration. Here we demonstrate that delivery of a constitutively active transcription factor that induces the production of endogenous IFNs and potentially other antiviral genes is a viable strategy to protect against FMD

Immunosuppression during Acute Infection with Foot-and-Mouth Disease Virus in Swine Is Mediated by IL-10

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal viruses, causing a devastating disease in cloven-hoofed animals with enormous economic consequences. Identification of the different parameters involved in the immune response elicited against FMDV remains unclear, and it is fundamental the understanding of such parameters before effective control measures can be put in place. In the present study, we show that interleukin-10 (IL-10) production by dendritic cells (DCs) is drastically increased during acute infection with FMDV in swine. In vitro blockade of IL-10 with a neutralizing antibody against porcine IL-10 restores T cell activation by DCs. Additionally, we describe that FMDV infects DC precursors and interferes with DC maturation and antigen presentation capacity. Thus, we propose a new mechanism of virus immunity in which a non-persistent virus, FMDV, induces immunosuppression by an increment in the production of IL-10, which in turn, reduces T cell function. This reduction of T cell activity may result in a more potent induction of neutralizing antibody responses, clearing the viral infection

Hidden Virulence Determinants in a Viral Quasispecies In Vivo▿

The characterization of virulence determinants of pathogenic agents is of utmost relevance for the design of disease control strategies. So far, two classes of virulence determinants have been characterized for viral populations: those imprinted in the nucleotide sequence of some specific genomic regions and those that depend on the complexity of the viral population as such. Here we provide evidence of a virulence determinant that depends neither on a genomic sequence nor on detectable differences in population complexity. Foot-and-mouth disease virus is lethal for C57BL/6 mice showing the highest viral load in pancreas. Virus isolated from pancreas after one passage in mice showed an attenuated phenotype, with no lethality even at the highest dose tested. By contrast, virus from sera of the same mice displayed a virulence similar to that of the parental wild-type clone and virus isolated from spleen displayed an intermediate phenotype. However, viral populations from pancreas, spleen, and serum showed indistinguishable consensus genomic nucleotide sequences and mutant spectrum complexities, as quantified according to the mutation frequencies of both entire genomic nucleotide sequences of biological clones. The results show that the populations with differing virulences cannot be distinguished either by the consensus sequence or by the average complexity of the mutant spectrum. Differential harvesting of virus generated by cell transfection of RNA from serum and pancreas failed to reveal genetic differences between subpopulations endowed with differing virulences. In addition to providing evidence of hidden virulence determinants, this study underlines the capacity of a clone of an RNA virus to rapidly diversify phenotypically in vivo

FMDV infection impairs T cell function.

<p>A. The allostimulatory capacity of uninfected MoDCs (triangles), FMDV C-S8c1-infected MoDCs at D0 (squares) and FMDV C-S8c1-infected MoDCs at D5 (circles) is indicated at different CD3⁺ T cells: MoDC ratios. Uninfected or infected MoDCs were irradiated and used as stimulators cells for allogeneic CD3⁺ T cells. Proliferation was measured in cpm (average cpm±SD) after [³H]Thymidine incorporation and are representative of three independent experiments. B. 72 hours after the onset of the co-cultures MoDCs and CD3⁺ T cells (ratio 1∶10), or MoDCs, supernatants were collected and analyzed for IFN-γ and IL-10 production by quantitative ELISA. The figure shows mean concentration values (pg/ml). Black bars: FMDV-infected MoDC at D0; white bars: FMDV-infected MoDCs at D5; gray bars: uninfected controls.</p

FMDV interferes with MoDC development <i>in vitro</i>.

<p>A. Dot plots show forward scatter (FSC) and side scatter (SSC) for uninfected, FMDV-infected at D0 and FMDV-infected at D5 before TNF-α stimulation (−TNF-α) and upon TNF-α treatment (+TNF-α). Note the lack of size increased in FMDV-infected MoDC after TNF-α at either D0 or D5 compared with uninfected control cells. It is shown the population with MoDCs phenotype in a circle and the percentage of this population is indicated. In the right panel, the viability and apoptosis of MoDCs analyzed by annexin-V and 7AAD staining is shown. This is the results from a representative experiment (n = 4) B. The mean fluorescence intensity (MFI) of surface molecules expressed on MoDCs. Each bar represents the MFI of a given surface molecule (CD80/86 or MHC class II) before and after TNF-α addition. Black bars: FMDV-infected MoDC at D0; white bars: FMDV-infected MoDC at D5; gray bars: uninfected cells. Data are average of four independent experiments±SD. Asterisks denote a statistically significant reduction in FMDV-infected MoDC compared with uninfected controls (student <i>t</i> test, p<0.05).</p

Anti-IL-10 restores T cell activation by MoDCs.

<p>A. The allostimulatory capacity of MoDCs from FMDV-infected swine (D1 to D17 pi) and naïve swine is evaluated in the presence of anti-IL-10 monoclonal Ab (gray squares) or an irrelevant antibody (white squares) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005659#s4" target="_blank">material and Methods</a>). MoDCs were irradiated and used as stimulator cells for allogeneic CD3⁺ T cells at the T cell/DC ratio indicated. Proliferation was measured in cpm after [³H]Thymidine incorporation. Data is representative of three independent experiments. B. IL-10 production by co-cultures of MoDC from FMDV-infected swine and T cells from a naïve pig. 72 h after the onset of the co-culture (MoDC: CD3⁺ T cell ratio of 1∶10), supernatants were collected and analyzed for IL-10 production by quantitative ELISA. The figure shows mean concentration values (pg/ml). C. IL-10 production by MoDCs from FMDV-infected swine. IL-10 production by cultures of MoDCs was determined by quantitative ELISA. It is expressed in pg/ml±SD. D. IL-10 produced in sera from FMDV-infected swine at different times post-inoculation. IL-10 was detected by ELISA. Each bar corresponds to one animal and it is expressed as pg/ml±SD. E. IFN-α produced in sera from FMDV-infected swine at days 1, 3, 5, 10 and 17 post-inoculation by ELISA. N, indicates naïve animals. Each bar corresponds to one animal and it is expressed as pg/ml±SD.</p

Replication of FMDV C-S8c1 in CD172⁺- MoDC.

<p>CD172⁺ cells were purified from swine PBMCs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005659#s4" target="_blank">Material and Methods</a>) and put in culture with rpGM-CSF and rpIL-4. The cells were infected either at D0 (just after purification) or D5 (5 days in culture with rpGM-CSF and rpIL-14) with FMDV C-S8c1 at moi of 10 PFU/cell. A. Staining of CD172⁺ cells infected at D0 with a monoclonal antibody against FMDV 3D (3H11) (green). At 24, 48 and 72 p.i. cells were fixed and analyzed by immunofluorescence microscopy. The percentage of positive cells for FMDV 3D in the field (average of at least 10 fields±SD) is indicated in the lower part of each panel. B. FMDV growth curve in CD172⁺ cells infected at D0. The results are representative of three independent experiments. C. Viral RNA expressed as the number of FMDV RNA molecules quantified by real time RT-PCR (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005659#s4" target="_blank">Material and Methods</a>) per 10⁴ cells. In the left column is indicated the day post-differentiation at which the viral RNA was extracted from FMDV-infected MoDCs. CD172⁺ D0 indicates CD172⁺ MoDCs infected with FMDV C-S8c1 at D0 post-differentiation; CD172⁺ D5 indicates CD172⁺ MoDCs infected at D5 post-differentiation. In the right column is indicated in braches the corresponding time post-infection. D. Confocal microscopy was used to analyze the expression of FMDV 3D protein (green) at 6 hpi of CD172⁺ cells infected with FMDV at D5 (5 days post-differentiation). Nuclei were stained with DAPI (blue). The micrographs are representative of at least three independent experiments.</p

MoDCs from FMDV-infected swine do not up-regulate CD80/86 and do not stimulate T cells.

<p>A. Flow cytometric analyses were performed to measure the expression of CD80/86 on MoDCs differentiated from PBMCs isolated from FMDV C-S8c1-infected swine. It is indicated the mean fluorescence intensity (MFI) of CD80/86 on MoDCs from naïve animals (N) and MoDCs from FMDV C-S8c1-infected pigs at different times post-inoculation (indicated as D1, D3, D5, D10 and D17) before treatment with TNF-α (black bars) and after treatment with TNF-α (white bars). B. The allostimulatory capacity of MoDCs isolated from FMDV-infected swine either treated with TNF-α (diamonds and dashed line) or untreated (squares) at different times post-infection (indicated in each graph). MoDCs were irradiated and used as stimulator cells for allogeneic CD3⁺ T cells at the T cell/DC ratio indicated. Proliferation was measured in cpm (average cpm±SD) after [³H]Thymidine incorporation. Data is representative of three independent experiments.</p

Selective Lymphocyte Depletion during the Early Stage of the Immune Response to Foot-and-Mouth Disease Virus Infection in Swine

Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious vesicular disease of cloven-hoofed animals. In the present study we use FMDV serotype C infection of swine to determine, by analytical techniques, the direct ex vivo visualization of virus-infected immune cells during the first 17 days of infection. We report, for the first time, that FMDV C-S8c1 can infect T and B cells at short periods of time postinoculation, corresponding with the peak of the viremia. There is a significant lymphopenia that involves CD3(+) CD4(−) CD8(+/−), CD3(+) CD4(−) CD8(+)Tc, and CD3(+) CD4(+) CD8(+) memory Th but not CD3(+) CD4(+) CD8(−) naïve Th lymphocytes. In addition, a profound depletion of the vast majority of peripheral T cells in lymph nodes and spleen is observed. This selective depletion of T cells is not due mainly to in situ death via apoptosis as visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) technique. Thus, early infection of T cells by FMDV may be the main cause of the observed T-cell depletion. Importantly, this lack of T cells is reflected in a reduced response to mitogen activation, which in many cases is totally eliminated. These data suggest a mechanism by which the virus causes a transient immunosuppression, subvert the immune systems, and spreads. These results have important implications for our understanding of early events in the development of a robust immune response against FMDV