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Foot-and-mouth disease (FMD) is a vesicular disease of cloven-hoofed animals with devastating economic implications. The current FMD vaccine, routinely used in enzootic countries, requires at least 7 days to induce protection. However, FMD vaccination is typically not recommended for use in non-enzootic areas, underscoring the need to develop new fast-acting therapies for FMD control during outbreaks. Interferons (IFNs) are among the immune system's first line of defense against viral infections. Bovine type III IFN delivered by a replication defective adenovirus (Ad) vector has effectively blocked FMD in cattle. However, the limited duration of protection--usually only 1-3 days post-treatment (dpt)--diminishes its utility as a field therapeutic. Here, we test whether polyethylene glycosylation (PEGylation) of recombinant bovine IFNλ3 (PEGboIFNλ3) can extend the duration of IFN-induced prevention of FMDV infection in both vaccinated and unvaccinated cattle. We treated groups of heifers with PEGboIFNλ3 alone or in combination with an adenovirus-based FMD O1Manisa vaccine (Adt-O1M) at either 3 or 5 days prior to challenge with homologous wild type FMDV. We found that pre-treatment with PEGboIFNλ3 was highly effective at preventing clinical FMD when administered at either time point, with or without co-administration of Adt-O1M vaccine. PEGboIFNλ3 protein was detectable systemically for >10 days and antiviral activity for 4 days following administration. Furthermore, in combination with Adt-O1M vaccine, we observed a strong induction of FMDV-specific IFNγ+ T cell response, demonstrating its adjuvanticity when co-administered with a vaccine. Our results demonstrate the promise of this modified IFN as a pre-exposure prophylactic therapy for use in emergency outbreak scenarios. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD), caused by the FMD virus (FMDV), is a highly contagious disease of cloven-hoofed livestock that can have severe economic impacts. Control and prevention strategies, including the development of improved vaccines, are urgently needed to effectively control FMD outbreaks in endemic settings. Previously, we employed two distinct strategies (codon pair bias deoptimization (CPD) and codon bias deoptimization (CD)) to deoptimize various regions of the FMDV serotype A subtype A12 genome, which resulted in the development of an attenuated virus in vitro and in vivo, inducing varying levels of humoral responses. In the current study, we examined the versatility of the system by using CPD applied to the P1 capsid coding region of FMDV serotype A subtype, A24, and another serotype, Asia1. Viruses carrying recoded P1 (A24-P1Deopt or Asia1-P1Deopt) exhibited different degrees of attenuation (i.e., delayed viral growth kinetics and replication) in cultured cells. Studies in vivo using a mouse model of FMD demonstrated that inoculation with the A24-P1Deopt and Asia1-P1Deopt strains elicited a strong humoral immune response capable of offering protection against challenge with homologous wildtype (WT) viruses. However, different results were obtained in pigs. While clear attenuation was detected for both the A24-P1Deopt and Asia1-P1Deopt strains, only a limited induction of adaptive immunity and protection against challenge was detected, depending on the inoculated dose and serotype deoptimized. Our work demonstrates that while CPD of the P1 coding region attenuates viral strains of multiple FMDV serotypes/subtypes, a thorough assessment of virulence and induction of adaptive immunity in the natural host is required in each case in order to finely adjust the degree of deoptimization required for attenuation without affecting the induction of protective adaptive immune responses. [ABSTRACT FROM AUTHOR]

Codon deoptimization (CD) has been recently used as a possible strategy to derive foot-and-mouth disease (FMD) live-attenuated vaccine (LAV) candidates containing DIVA markers. However, reversion to virulence, or loss of DIVA, from possible recombination with wild-type (WT) strains has yet to be analyzed. An in vitro assay was developed to quantitate the levels of recombination between WT and a prospective A24-P2P3 partially deoptimized LAV candidate. By using two genetically engineered non-infectious RNA templates, we demonstrate that recombination can occur within non-deoptimized viral genomic regions (i.e., 3′end of P3 region). The sequencing of single plaque recombinants revealed a variety of genome compositions, including full-length WT sequences at the consensus level and deoptimized sequences at the sub-consensus/consensus level within the 3′end of the P3 region. Notably, after further passage, two recombinants that contained deoptimized sequences evolved to WT. Overall, recombinants featuring large stretches of CD or DIVA markers were less fit than WT viruses. Our results indicate that the developed assay is a powerful tool to evaluate the recombination of FMDV genomes in vitro and should contribute to the improved design of FMDV codon deoptimized LAV candidates. [ABSTRACT FROM AUTHOR]

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus–host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are considered the first line of defense against viral diseases. Due to their ability to modulate immune responses, they have become an attractive therapeutic option to control virus infections. In fact, like many other viruses, foot-and-mouth disease virus (FMDV), the most contagious pathogen of cloven-hoofed animals, is highly sensitive to the action of IFNs. Previous studies demonstrated that type I, II, and III IFNs, expressed using a replication defective human adenovirus 5 (Ad5) vector, can effectively block FMDV replication in vitro and can protect animals when challenged 1 day after Ad5-IFN treatment, in some cases providing sterile immunity. Rapidly spreading foot-and-mouth disease (FMD) is currently controlled with vaccination, although development of a protective adaptive immune response takes 5–7 days. Therefore, an optimal strategy to control FMD outbreaks is to block virus replication and spread through sustained IFN activity while the vaccine-stimulated adaptive immune response is developed. Challenges with methods of delivery and/or with the relative short IFN protein half-life in vivo , have halted the development of such approach to effectively control FMD in the animal host. One strategy to chemically improve drug pharmacodynamics is the use of pegylation. In this proof-of-concept study, we demonstrate that pegylated recombinant porcine (po)IFNα displays strong and long-lasting antiviral activity against FMDV in vitro and in vivo , completely protecting swine against FMD for at least five days after a single dose. These results highlight the potential of this biotherapeutics to use in combination with vaccines to fully control FMD in the field. [ABSTRACT FROM AUTHOR]

The type I interferon response is an important innate antiviral pathway. Recognition of viral RNA by RIG-I-like receptors (RLRs) activates a signaling cascade that leads to type I interferon (IFN-α/β) gene transcription. Multiple proteins in this signaling pathway (e.g. RIG-I, MDA5, MAVS, TBK1, IRF3) are regulated by (de)ubiquitination events. Most viruses have evolved mechanisms to counter this antiviral response. The leader protease (Lpro) of foot-and-mouth-disease virus (FMDV) has been recognized to reduce IFN-α/β gene transcription; however, the exact mechanism is unknown. The proteolytic activity of Lpro is vital for releasing itself from the viral polyprotein and for cleaving and degrading specific host cell proteins, such as eIF4G and NF-κB. In addition, Lpro has been demonstrated to have deubiquitination/deISGylation activity. Lpro's deubiquitination/deISGylation activity and the cleavage/degradation of signaling proteins have both been postulated to be important for reduced IFN-α/β gene transcription. Here, we demonstrate that TBK1, the kinase that phosphorylates and activates the transcription factor IRF3, is cleaved by Lpro in FMDV-infected cells as well as in cells infected with a recombinant EMCV expressing Lpro. In vitro cleavage experiments revealed that Lpro cleaves TBK1 at residues 692–694. We also observed cleavage of MAVS in HeLa cells infected with EMCV-Lpro, but only observed decreasing levels of MAVS in FMDV-infected porcine LFPK αVβ6 cells. We set out to dissect Lpro's ability to cleave RLR signaling proteins from its deubiquitination/deISGylation activity, to determine their relative contributions to the reduction of IFN-α/β gene transcription. The introduction of specific mutations, of which several were based on the recently published structure of Lpro in complex with ISG15, allowed us to identify specific amino acid substitutions that separate the different proteolytic activities of Lpro. Characterization of the effects of these mutations revealed that Lpro's ability to cleave RLR signaling proteins but not its deubiquitination/deISGylation activity correlates with the reduced IFN-β gene transcription. Author summary: Outbreaks of the picornavirus foot-and-mouth disease virus (FMDV) have significant consequences for animal health and product safety and place a major economic burden on the global livestock industry. Understanding how this notorious animal pathogen suppresses the antiviral type I interferon (IFN-α/β) response may help to develop countermeasures to control FMDV infections. FMDV suppresses the IFN-α/β response through the activity of its Leader protein (Lpro), a protease that can cleave host cell proteins. Lpro was also shown to have deubiquitinase and deISGylase activity, raising the possibility that Lpro suppresses IFN-α/β by removing ubiquitin and/or ISG15, two posttranslational modifications that can regulate the activation, interactions and localization of (signaling) proteins. Here, we show that TBK1 and MAVS, two signaling proteins that are important for activation of IFN-α/β gene transcription, are cleaved by Lpro. By generating Lpro mutants lacking either of these two activities, we demonstrate that Lpro's ability to cleave signaling proteins, but not its deubiquitination/deISGylase activity, correlates with suppression of IFN-β gene transcription. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. The disease affects many areas of the world, often causing extensive epizootics in livestock, mostly farmed cattle and swine, although sheep, goats and many wild species are also susceptible. In countries where food and farm animals are essential for subsistence agriculture, outbreaks of FMD seriously impact food security and development. In highly industrialized developed nations, FMD endemics cause economic and social devastation mainly due to observance of health measures adopted from the World Organization for Animal Health (OIE). High morbidity, complex host-range and broad genetic diversity make FMD prevention and control exceptionally challenging. In this article we review multiple vaccine approaches developed over the years ultimately aimed to successfully control and eradicate this feared disease. [ABSTRACT FROM AUTHOR]

The genetic regulatory network controlling the innate immune system is well understood in many species. However, the role of the epigenetic mechanisms underlying the expression of immunoregulatory genes is less clear, especially in livestock species. Histone H3 lysine 9 dimethylation (H3K9me2) is an epigenetic modification associated with transcriptional silencing within the euchromatin regions. Euchromatic histone-lysine N-methyltransferase 2 (EHMT2; also known as G9a) is a crucial enzyme responsible for regulating the dynamics of this epigenetic modification. It has been shown that histone modifications play a role in regulating type I interferon (IFN) response. In the present study, we investigated the role of EHMT2 in the epigenetic regulation of bovine antiviral innate immunity and explored its therapeutic potential against viral infections. We evaluated the effects of pharmacological and RNAi-mediated inhibition of EHMT2 on the transcription of IFN-β and other IFN-inducible antiviral genes, as well as its effect on foot-and-mouth disease virus (FMDV) and vesicular stomatitis virus (VSV) replication in bovine cells. We show that treatment of primary bovine cells with the synthetic EHMT2 inhibitor (UNC0638) either before or shortly after virus infection resulted in a significant increase in transcript levels of bovine IFN-β ( boIFN-β; 300-fold) and other IFN-inducible genes, including IFN-stimulated gene 15 (ISG-15), myxovirus resistance 1 ( Mx-1), Mx-2, RIG-I, 2′,5′-oligoadenylate synthetase 1 ( OAS-1), and protein kinase R ( PKR). Expression of these factors correlated with a significant decrease in VSV and FMDV viral titers. Our data confirm the involvement of EHMT2 in the epigenetic regulation of boIFN-β and demonstrate the activation of a general antiviral state after EHMT2 inhibition. [ABSTRACT FROM AUTHOR]

The leader proteinase (Lpro) 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-β mRNA and IFN-stimulated genes. In addition to its role in shutting off cap-dependent host mRNA translation, Lpro is associated with the degradation of the p65/RelA subunit of nuclear factor κB (NF-κB). Bioinformatics analysis suggests that Lpro 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 Lpro. Although three stable mutant viruses were obtained, only the double mutant displayed an attenuated phenotype in cell culture. Indirect immunofluorescence analysis showed that Lpro 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-κB-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 Lpro 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 Lpro. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of cloven-hoofed animals. In this review, we discuss the mechanisms FMDV has evolved to counteract the host innate and adaptive immune responses and the role of viral proteins in this process. The viral leader proteinase, Lpro, limits the host innate response by inhibiting the induction of interferon beta (IFNβ) mRNA and blocking host cell translation. A second viral proteinase, 3Cpro, may affect host cell transcription because it cleaves histone H3. Viral protein 2B in conjunction with 2C or their precursor 2BC inhibits protein trafficking through the endoplasmic reticulum and Golgi apparatus. A decrease in surface expression of major histocompatibility class I molecules during FMDV infection suggests that 2B, 2C and/or 2BC may be involved in delaying the initiation of the host adaptive immune response and also adversely affect the secretion of induced signaling molecules. FMDV also causes a transient lymphopenia in swine, but the mechanism involved is not understood nor have any viral protein(s) been implicated. Furthermore, the interaction of FMDV with various cells in the immune system including lymphocytes and dendritic cells and the possible role of apoptosis and autophagy in these interactions are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Foot-and-mouth disease virus (FMDV) is one of the most contagious agents of animals. Recent disease outbreaks in FMD-free countries have prompted the development of new control strategies that could improve the levels of protection against this virus. We have delivered a plasmid expressing a short hairpin RNA (shRNA) directed against a highly conserved sequence in the 2B nonstructural protein coding region of FMDV RNA to porcine cells. After virus infection, these cells showed a significant reduction in the synthesis of viral RNA and proteins, as well as a decrease in virus yield when compared to control cells. The antiviral effect was sequence specific and not attributable to induction of the interferon pathway. Since FMDV is an antigenically variable virus, we examined the effectiveness of this strategy against multiple serotypes and found that expressed 2B shRNA resulted in efficient silencing of at least 4 FMDV serotypes. Thus, RNA interference may be a potential alternative control strategy to limit the spread of this highly contagious virus in livestock. [Copyright &y& Elsevier]

RNA interference (RNAi) is a mechanism of gene silencing that is being examined as an antiviral strategy in mammalian cells. Chen et al. have used this approach to examine its potential against foot-and-mouth disease (FMD), an extremely contagious viral disease of cloven-hoofed animals. This Research Focus discusses these experiments and comments on the possible use of RNAi, as well as other antiviral strategies, as a rapid method of FMD control. [Copyright &y& Elsevier]

Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD. IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD. [ABSTRACT FROM AUTHOR]

Foot and Mouth Disease is a highly contagious and economically important disease of livestock. While vaccination is often effective at controlling viral spread, failures can occur due to strain mismatch or viral mutation. Foot and Mouth Disease Virus (FMDV) possesses a hypervariable region within the G-H Loop of VP1, a capsid protein commonly associated with virus neutralization. Here, we investigate the effect of replacement of the G-H loop hypervariable epitope with a xenoepitope from PRRS virus on the immunogenicity and efficacy of an adenovirus vectored FMDV vaccine (Ad5-FMD). Pigs were vaccinated with Ad5-FMD, the modified Ad5-FMD xeno , or PBS, followed by intradermal challenge with FDMV strain O 1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMD xeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMD xeno , when compared to those vaccinated with Ad5-FMD, prior to viral challenge, indicative of immune redirection away from VP1 towards non-neutralizing epitopes. As expected, animals vaccinated with unmodified Ad5-FMD were protected from lesions, fever, and viremia. In contrast, animals vaccinated with Ad5-FMD xeno developed clinical signs and viremia, but at lower levels than that observed in PBS-treated controls. No significant difference was found in nasal shedding of virions between the two Ad5-FMD vaccinated groups. This data suggests that the hypervariable epitope of the VP1 G-H loop contributes to protective immunity conferred by Ad5 vector-delivered FMD vaccines in swine, and cannot be substituted without a loss of immunogenicity. [ABSTRACT FROM AUTHOR]

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV, genus aphthovirus), one of the most notorious pathogens in global livestock industry, needs to navigate antivirals host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I IFN response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV Lpro or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2, but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved amongst aphthoviruses. Neither FMDV or ERAV Lpro interfered with phosphorylation of PKR or eIF2α, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response. [ABSTRACT FROM AUTHOR]

The S fragment of the FMDV 5’ UTR is predicted to fold into a long stem-loop structure and it has been implicated in virus-host protein interactions. In this study, we report the minimal S fragment sequence required for virus viability and show a direct correlation between the extent of the S fragment deletion mutations and attenuated phenotypes. Furthermore, we provide novel insight into the role of the S fragment in modulating the host innate immune response. Importantly, in an FMDV mouse model system, all animals survive the inoculation with the live A 24 FMDV-S 4 mutant, containing a 164 nucleotide deletion in the upper S fragment loop, at a dose 1000 higher than the one causing lethality by parental A 24 FMDV, indicating that the A 24 FMDV-S 4 virus is highly attenuated in vivo . Additionally, mice exposed to high doses of live A 24 FMDV-S 4 virus are fully protected when challenged with parental A 24 FMDV virus. [ABSTRACT FROM AUTHOR]

The foot-and-mouth disease virus (FMDV) leader protease (L pro ) inhibits host translation and transcription affecting the expression of several factors involved in innate immunity. In this study, we have identified the host transcription factor ADNP (activity dependent neuroprotective protein) as an L pro interacting protein by mass spectrometry. We show that L pro can bind to ADNP in vitro and in cell culture. RNAi of ADNP negatively affected virus replication and higher levels of interferon (IFN) and IFN-stimulated gene expression were detected. Importantly, infection with FMDV wild type but not with a virus lacking Lpro (leaderless), induced recruitment of ADNP to IFN-α promoter sites early during infection. Furthermore, we found that L pro and ADNP are in a protein complex with the ubiquitous chromatin remodeling factor Brg-1. Our results uncover a novel role of FMDV L pro in targeting ADNP and modulation of its transcription repressive function to decrease the expression of IFN and ISGs. [ABSTRACT FROM AUTHOR]

Sam68 was previously shown to be a critical host factor for foot-and-mouth disease virus (FMDV) replication. MicroRNA (miR) miR-203a is reportedly a negative regulator of Sam68 expression both in vitro and in vivo . Here, transfection of miR-203a-3p and miR-203a-5p mimics separately and in combination in a porcine cell line followed by FMDV infection resulted in diminished viral protein synthesis and a 4 and 6 log reduction in virus titers relative to negative controls, respectively. Unexpectedly, Sam68 expression was increased by miR-203a-5p transfection, but not miR-203a-3p. miR-203a-5p also down-regulated Survivin expression, which was predicted to play a role in FMDV infection. Moreover, miR-203a-5p but not miR-203a-3p affected a reduction in FMDV viral RNA. These effects were not replicated with a related Picornavirus, suggesting FMDV specificity. Importantly, miR-203a-3p and miR-203a-5p impaired FMDV infection across multiple FMDV serotypes. We concluded that miR-203a-3p and miR-203a-5p represent attractive potential naturally occurring bio-therapeutics against FMDV. [ABSTRACT FROM AUTHOR]

A human adenovirus (Ad5) vectored foot-and-mouth disease virus (FMDV) O1-Manisa subunit vaccine (Ad5-O1Man) was engineered to deliver FMDV O1-Manisa capsid and capsid-processing proteins. Swine inoculated with Ad5-O1Man developed an FMDV-specific humoral response as compared to animals inoculated with an empty Ad5-vector. Vaccinated animals were completely protected against homologous challenge at 7 or 21 days post-vaccination. Potency studies exhibited a PD50 of about 10 7 pfu/animal while a dose of 4×10 7 pfu/animal fully protected swine against FMDV intradermal challenge. In-vitro cross-neutralization analysis distinctly predicted that swine vaccinated with Ad5-O1Man would be protected against challenge with homologous FMDV O1Man Middle East-South Asia (ME-SA) topotype and also against recent outbreak strains of Mya-98 South East Asia (SEA) lineage including O1-UK-2001 and O1-South Korea-2010. These results indicate that recombinant Ad5-O1Man is an effective, safe and cross-reacting vaccine that could potentially be used preventively and in outbreak situations, to control FMDV O Mya-98 lineage in swine. [ABSTRACT FROM AUTHOR]

Foot-and-mouth-disease (FMD) remains the most infectious livestock disease worldwide. Although commercially available inactivated or adenovirus-vectored-vaccines (Ad5-FMD) are effective, they require 5–7 days to induce protection. Therefore, new control strategies that stimulate rapid immune responses are needed. Expression of bovine interferon λ3 using the Ad5-vector platform (Ad5-boIFNλ3) is able to delay disease in cattle, but clinical signs appear at 9 days after challenge. We hypothesized that combination of Ad5-boIFNλ3 and Ad5-FMD could induce immediate and lasting protection against FMD. Cattle were vaccinated with an Ad5-FMD, Ad5-boIFNλ3, or the combination of both, followed by challenge at three days post-immunization. All animals treated with Ad5-FMD combined with Ad5-boIFNλ3 were fully protected against FMD, despite the absence of systemic neutralizing antibodies or antiviral activity at the time of challenge. Induction of a strong cell-mediated immune response suggested that Ad5-boIFNλ3 is able to act as an adjuvant of Ad5-FMD vaccine in cattle. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) remains one of the most devastating livestock diseases around the world. Several serotype-specific vaccine formulations exist, but they require about 5 to 7 days to induce protective immunity. Our previous studies have shown that a constitutively active fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 [IRF7/3(5D)] strongly induced type I IFN and antiviral genes in vitro and prevented mortality in an FMD mouse model when delivered with a replication-defective adenoviral vector [Ad5-poIRF7/3(5D)]. Here, we demonstrate that pigs treated with 108, 109, or 1010 PFU of Ad5-poIRF7/3(5D) 24 h before FMDV challenge were fully protected from FMD clinical signs and did not develop viremia, virus shedding or antibodies against FMDV nonstructural proteins. Pigs treated with Ad5-poIRF7/3(5D) had higher levels of IFN and antiviral activity in serum, and upregulated expression of several IFN-stimulated genes in peripheral blood mononuclear cells, compared to pigs treated with Ad5-Blue vector control. Importantly, treatment of porcine cultured cells with Ad5-poIRF7/3(5D) inhibited the replication of all 7 FMDV serotypes. In vitro experiments using cultured embryonic fibroblasts derived from IFN receptor knockout mice suggested that the antiviral response induced by Ad5-poIRF7/3(5D) was dependent on type I and III IFN pathways; however, experiments with mice demonstrated that a functional type I IFN pathway mediates Ad5-poIRF7/3(5D) protection conferred in vivo. Our studies demonstrate that inoculation with Ad5-poIRF7/3(5D) completely protects swine against FMD by inducing a strong type I IFN response and highlights its potential application to rapidly and effectively prevent FMDV replication and dissemination. [ABSTRACT FROM AUTHOR]

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co 2+ affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni 2+ -chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance. [ABSTRACT FROM AUTHOR]

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 log10 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. [ABSTRACT FROM AUTHOR]

Abstract: Early events in the pathogenesis of foot-and-mouth disease virus (FMDV) infection in cattle were investigated through aerosol and intraepithelial lingual (IEL) inoculations of a cDNA-derived FMDV-A24 wild type virus (FMDV-WT) or a mutant derived from the same clone (FMDV-Mut). After aerosolization of FMDV-WT, primary infection sites had significantly greater quantities of FMDV, viral RNA, and type I/III interferon (IFN) activity compared to corresponding tissues from cattle infected with FMDV-Mut. Additionally, FMDV-WT-infected cattle had marked induction of systemic IFN activity in serum. In contrast, FMDV-Mut aerosol-infected cattle did not manifest systemic IFN response nor had viremia. Interestingly, IEL inoculation of FMDV-Mut in cattle restored the virulent phenotype and systemic IFN response. These data indicate that the attenuated phenotype in cattle is associated with decreased replicative efficiency, reflected by decreased innate response. However, attenuation is abrogated by bypassing the common primary infection sites, inducing accelerated viral replication at the inoculation site. [Copyright &y& Elsevier]

We have previously shown that delivery of the porcine type I interferon gene (poIFN-ɑ/β) 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-ɑ (VRP-poIFN-ɑ) 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 107 or 108 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-ɑ. 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-ɑ and VRP-GFP, is directly involved in protection against FMDV. [ABSTRACT FROM AUTHOR]

Interferons (IFNs) are the first line of defense against viral infections. Although type I and II IFNs have proven effective to inhibit foot-and-mouth disease virus (FMDV) replication in swine, a similar approach had only limited efficacy in cattle. Recently, a new family of IFNs, type III IFN or IFN-?, has been identified in human, mouse, chicken, and swine. We have identified bovine IFN-?3 (boIFN-?3), also known as interleukin 28B (IL-28B), and demonstrated that expression of this molecule using a recombinant replication-defective human adenovirus type 5 (Ad5) vector, Ad5-boIFN-?3, exhibited antiviral activity against FMDV in bovine cell culture. Furthermore, inoculation of cattle with Ad5-boIFN-?3 induced systemic antiviral activity and upregulation of IFN-stimulated gene expression in the upper respiratory airways and skin. In the present study, we demonstrated that disease could be delayed for at least 6 days when cattle were inoculated with Ad5-boIFN-?3 and challenged 24 h later by intradermolingual inoculation with FMDV. Furthermore, the delay in the appearance of disease was significantly prolonged when treated cattle were challenged by aerosolization of FMDV, using a method that resembles the natural route of infection. No clinical signs of FMD, viremia, or viral shedding in nasal swabs was found in the Ad5-boIFN-?3-treated animals for at least 9 days postchallenge. Our results indicate that boIFN-?3 plays a critical role in the innate immune response of cattle against FMDV. To this end, this work represents the most successful biotherapeutic strategy so far tested to control FMDV in cattle. [ABSTRACT FROM AUTHOR]