Venezuelan equine encephalitis replicon particles can induce rapid protection against foot-and-mouth disease virus.

We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/ß) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 10(7) or 10(8) infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV.

Porcine type I interferon rapidly protects swine against challenge with multiple serotypes of foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Current inactivated vaccines require approximately 7 days to induce protection, but before this time vaccinated animals remain susceptible to disease. Previously, we demonstrated that intramuscular (IM) inoculation of a replication-defective human adenovirus type 5 (Ad5) vector containing a porcine interferon α gene (pIFNα) can protect swine challenged 1 day later by intradermal (ID) injection with FMDV A24 Cruzeiro from both clinical disease and virus replication. To extend these studies to other FMDV serotypes, we demonstrated the effectiveness of Ad5-pIFNα against ID challenge with O1 Manisa and Asia-1 and against A24 Cruzeiro in a direct contact challenge model. We also showed that an Ad5 vector containing the pIFNß gene can protect swine against ID challenge with A24 Cruzeiro. Further, IM inoculation of a 10-fold lower dose of Ad5-pIFNα at 4 sites in the neck compared with 1 site in the hind limb can protect swine against ID challenge. These studies demonstrate the ability of Ad5-delivered type I IFN to rapidly protect swine against several FMDV serotypes and suggest that various modifications of this approach may enable this strategy to be successfully used in other FMD susceptible species.

Interferon-induced protection against foot-and-mouth disease virus infection correlates with enhanced tissue-specific innate immune cell infiltration and interferon-stimulated gene expression.

Previously, we demonstrated that type I interferon (IFN-alpha/beta) or a combination of IFN-alpha/beta and type II IFN (IFN-gamma) delivered by a replication-defective human adenovirus 5 (Ad5) vector protected swine when challenged 1 day later with foot-and-mouth disease virus (FMDV). To gain a more comprehensive understanding of the mechanism of protection induced by IFNs, we inoculated groups of six swine with Ad5-vectors containing these genes, challenged 1 day later and euthanized 2 animals from each group prior to (1 day postinoculation [dpi]) and at 1 (2 dpi) and 6 days postchallenge (7 dpi). Blood, skin, and lymphoid tissues were examined for IFN-stimulated gene (ISG) induction and infiltration by innate immune cells. All IFN-inoculated animals had delayed and decreased clinical signs and viremia compared to the controls, and one animal in the IFN-alpha treated group did not develop disease. At 1 and 2 dpi the groups inoculated with the IFNs had increased numbers of dendritic cells and natural killer cells in the skin and lymph nodes, respectively, as well as increased levels of several ISGs compared to the controls. In particular, all tissues examined from IFN-treated groups had significant upregulation of the chemokine 10-kDa IFN-gamma-inducible protein 10, and preferential upregulation of 2',5'-oligoadenylate synthetase, Mx1, and indoleamine 2,3-dioxygenase. There was also upregulation of monocyte chemotactic protein 1 and macrophage inflammatory protein 3alpha in the skin. These data suggest that there is a complex interplay between IFN-induced immunomodulatory and antiviral activities in protection of swine against FMDV.

A conserved domain in the leader proteinase of foot-and-mouth disease virus is required for proper subcellular localization and function.

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) is involved in antagonizing the innate immune response by blocking the expression of interferon (IFN) and by reducing the immediate-early induction of IFN-beta mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, L(pro) is associated with the degradation of the p65/RelA subunit of nuclear factor kappaB (NF-kappaB). Bioinformatics analysis suggests that L(pro) contains a SAP (for SAF-A/B, Acinus, and PIAS) domain, a protein structure associated in some cases with the nuclear retention of molecules involved in transcriptional control. We have introduced a single or a double mutation in conserved amino acid residues contained within this domain of L(pro). Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that L(pro) subcellular distribution is altered in cells infected with the double mutant virus. Interestingly, nuclear p65/RelA staining disappeared from wild-type (WT) FMDV-infected cells but not from double mutant virus-infected cells. Consistent with these results, NF-kappaB-dependent transcription was not inhibited in cells infected with double mutant virus in contrast to cells infected with WT virus. However, degradation of the translation initiation factor eIF-4G was very similar for both the WT and the double mutant viruses. Since L(pro) catalytic activity was demonstrated to be a requirement for p65/RelA degradation, our results indicate that mutation of the SAP domain reveals a novel separation-of-function activity for FMDV L(pro).