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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.

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.

Adenovirus vectors offer a convenient platform for the expression of antigens and have become an attractive system for vaccine development. Currently, the most successful approach to the development of new foot-and-mouth disease (FMD) vaccines has been the production of a replication-defective human serotype 5 adenovirus that delivers the capsid and capsid processing coding regions of FMD virus (FMDV) (Ad5-FMD). A specific construct for FMDV serotype A24 has been fully developed into a commercial product fulfilling the requirements of the Center of Veterinary Biologics (CVB) of the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture (USDA), for commercialization in the USA. In this chapter, we describe a standard protocol for the generation and small-scale production of Ad5-FMDV serotype O1Manisa vaccines. We use directional cloning to introduce the FMDV O1Manisa capsid in the Ad5-Blue vector. This is followed by the linearization of the recombinant Ad5 with Pac I and transfection into HEK293 cells for rescue and propagation, and then by increased production and purification. Finally, purified recombinant virus is characterized by determining virus yield and expression of targeted antigen in specific cell type of interest., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: MAL is now employed by MSD animal health, his involvement in this study was unrelated to his position at MSD.

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., (Copyright © 2020 Medina et al.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus ), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (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 leader protease (L pro ) or 3C pro , we demonstrate an essential role for L pro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking L pro resulted in SG formation. Additionally, we show that L pro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) L pro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV L pro interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2α, indicating that L pro 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. IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (L pro ). Simultaneously, we observed that L pro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future., (Copyright © 2019 American Society for Microbiology.)

Foot-and-mouth disease (FMD) continues to be the viral disease posing the greatest economic threat to agriculture. An unusually fast replication rate, extreme transmissibility, broad species tropism and antigenic diversity have made its etiologic agent, FMD virus, a difficult pathogen to defeat. Over the last 70 years, use of an inactivated virus vaccine has played a key role in disease control and eradication was possible in certain regions of the world. However, a rapidly changing environment, increased trade, population growth, international travel and migration, contribute to disease resurgence, challenging the capabilities of any available vaccine. Here we review the current knowledge on FMD vaccines and provide an outlook of novel technologies as possible improved alternatives for disease control and eradication., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Published by Elsevier Inc.)

Foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase (RdRp) (3D pol ) catalyzes viral RNA synthesis. Its characteristic low fidelity and absence of proofreading activity allow FMDV to rapidly mutate and adapt to dynamic environments. In this study, we used the structure of FMDV 3D pol in combination with previously reported results from similar picornaviral polymerases to design point mutations that would alter replication fidelity. In particular, we targeted Trp237 within conserved polymerase motif A because of the low reversion potential inherent in the single UGG codon. Using biochemical and genetic tools, we show that the replacement of tryptophan 237 with phenylalanine imparts higher fidelity, but replacements with isoleucine and leucine resulted in lower-fidelity phenotypes. Viruses containing these W237 substitutions show in vitro growth kinetics and plaque morphologies similar to those of the wild-type (WT) A 24 Cruzeiro strain in BHK cells, and both high- and low-fidelity variants retained fitness during coinfection with the wild-type virus. The higher-fidelity W237F (W237F HF ) mutant virus was more resistant to the mutagenic nucleoside analogs ribavirin and 5-fluorouracil than the WT virus, whereas the lower-fidelity W237I (W237I LF ) and W237L LF mutant viruses exhibited lower ribavirin resistance. Interestingly, the variant viruses showed heterogeneous and slightly delayed growth kinetics in primary porcine kidney cells, and they were significantly attenuated in mouse infection experiments. These data demonstrate, for a single virus, that either increased or decreased RdRp fidelity attenuates virus growth in animals, which is a desirable feature for the development of safer and genetically more stable vaccine candidates. IMPORTANCE Foot-and-mouth disease (FMD) is the most devastating disease affecting livestock worldwide. Here, using structural and biochemical analyses, we have identified FMDV 3D pol mutations that affect polymerase fidelity. Recombinant FMDVs containing substitutions at 3D pol tryptophan residue 237 were genetically stable and displayed plaque phenotypes and growth kinetics similar to those of the wild-type virus in cell culture. We further demonstrate that viruses harboring either a W237F HF substitution or W237I LF and W237L LF mutations were highly attenuated in animals. Our study shows that obtaining 3D pol fidelity variants by protein engineering based on polymerase structure and function could be exploited for the development of attenuated FMDV vaccine candidates that are safer and more stable than strains obtained by selective pressure via mutagenic nucleotides or adaptation approaches., (Copyright © 2017 American Society for Microbiology.)

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., (Published by Elsevier B.V.)

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., (Copyright © 2017. Published by Elsevier Inc.)

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 6log 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., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

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., (Published by Elsevier Inc.)

Unlabelled: 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 10(8), 10(9), or 10(10) 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., Importance: Foot-and-mouth disease virus (FMDV) causes a fast-spreading disease that affects farm animals, with economically and socially devastating consequences. Our study shows that inoculation with a constitutively active transcription factor, namely, a fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 delivered by an adenovirus vector [Ad5-poIRF7/3(5D)], is a new effective treatment to prevent FMD in swine. Animals pretreated with Ad5-poIRF7/3(5D) 1 day before being exposed to FMDV were completely protected from viral replication and clinical disease. It is noteworthy that the doses of Ad5-poIRF7/3(5D) required for protection are lower than those previously reported for similar approaches using Ad5 vectors delivering type I, II, or III IFN, suggesting that this novel strategy would be economically appealing to counteract FMD. Our results also indicate that a dynamic interplay among different components of pigs' innate immune defenses allows potent antiviral effects after Ad5-poIF7/3(5D) administration., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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., (Published by Elsevier Inc.)

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.

Novel vaccination approaches against foot-and-mouth disease (FMD) include the use of replication-defective human adenovirus type 5 (Ad5) vectors that contain the capsid-encoding regions of FMD virus (FMDV). Ad5 containing serotype A24 capsid sequences (Ad5.A24) has proved to be effective as a vaccine against FMD in livestock species. However, Ad5-vectored FMDV serotype O1 Campos vaccine (Ad5.O1C.2B) provides only partial protection of cattle against homologous challenge. It has been reported that a fiber-modified Ad5 vector expressing Arg-Gly-Asp (RGD) enhances transduction of antigen-presenting cells (APC) in mice. In the current study, we assessed the efficacy of a fiber-modified Ad5 (Adt.O1C.2B.RGD) in cattle. Expression of FMDV capsid proteins was superior in cultured cells infected with the RGD-modified vector. Furthermore, transgene expression of Adt.O1C.2B.RGD was enhanced in cell lines that constitutively express integrin αvβ6, a known receptor for FMDV. In contrast, capsid expression in cattle-derived enriched APC populations was not enhanced by infection with this vector. Our data showed that vaccination with the two vectors yielded similar levels of protection against FMD in cattle. Although none of the vaccinated animals had detectable viremia, FMDV RNA was detected in serum samples from animals with clinical signs. Interestingly, CD4(+) and CD8(+) gamma interferon (IFN-γ)(+) cell responses were detected at significantly higher levels in animals vaccinated with Adt.O1C.2B.RGD than in animals vaccinated with Ad5.O1C.2B. Our results suggest that inclusion of an RGD motif in the fiber of Ad5-vectored FMD vaccine improves transgene delivery and cell-mediated immunity but does not significantly enhance vaccine performance in cattle., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Codon bias deoptimization has been previously used to successfully attenuate human pathogens, including poliovirus, respiratory syncytial virus, and influenza virus. We have applied a similar technology to deoptimize the capsid-coding region (P1) of foot-and-mouth disease virus (FMDV). Despite the introduction of 489 nucleotide changes (19%), synonymous deoptimization of the P1 region rendered a viable FMDV progeny. The resulting strain was stable and reached cell culture titers similar to those obtained for wild-type (WT) virus, but at reduced specific infectivity. Studies in mice showed that 100% of animals inoculated with the FMDV A12 P1 deoptimized mutant (A12-P1 deopt) survived, even when the animals were infected at doses 100 times higher than the dose required to cause death by WT virus. All mice inoculated with the A12-P1 deopt mutant developed a strong antibody response and were protected against subsequent lethal challenge with WT virus at 21 days postinoculation. Remarkably, the vaccine safety margin was at least 1,000-fold higher for A12-P1 deopt than for WT virus. Similar patterns of attenuation were observed in swine, in which animals inoculated with A12-P1 deopt virus did not develop clinical disease until doses reached 1,000 to 10,000 times the dose required to cause severe disease in 2 days with WT A12. Consistently, high levels of antibody titers were induced, even at the lowest dose tested. These results highlight the potential use of synonymous codon pair deoptimization as a strategy to safely attenuate FMDV and further develop live attenuated vaccine candidates to control such a feared livestock disease., Importance: Foot-and-mouth disease (FMD) is one of the most feared viral diseases that can affect livestock. Although this disease appeared to be contained in developed nations by the end of the last century, recent outbreaks in Europe, Japan, Taiwan, South Korea, etc., have demonstrated that infection can spread rapidly, causing devastating economic and social consequences. The Global Foot-and-Mouth Disease Research Alliance (GFRA), an international organization launched in 2003, has set as part of their five main goals the development of next-generation control measures and strategies, including improved vaccines and biotherapeutics. Our work demonstrates that newly developed codon pair bias deoptimization technologies can be applied to FMD virus to obtain attenuated strains with potential for further development as novel live attenuated vaccine candidates that may rapidly control disease without reverting to virulence., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: 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., 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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Published by Elsevier Inc.)

We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/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-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.

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. Vaccines require ∼7 days to induce protection; thus, before this time, vaccinated animals are still susceptible to the disease. Our group has previously shown that swine inoculated with 1×10(11) focus forming units (FFU) of a replication-defective human adenovirus containing the gene for porcine interferon alpha (Adt-pIFN-α) are sterilely protected from FMDV serotypes A24, O1 Manisa, or Asia 1 when the animals are challenged 1 day postadministration, and protection can last for 3-5 days. Polyriboinosinic-polyribocytidylic acid stabilized with poly-l-lysine and carboxymethyl cellulose (poly ICLC) is a synthetic double-stranded RNA that is a viral mimic and activates multiple innate immune pathways through interaction with toll-like receptor 3 and MDA-5. It is a potent inducer of IFNs. In this study, we initially examined the effect of poly IC and IFN-α on FMDV replication and gene induction in cell culture. Poly ICLC alone or combined with Adt-pIFN-α was then evaluated for its therapeutic efficacy in swine against intradermal challenge with FMDV A24, 1 day post-treatment. Groups of swine were subcutaneously inoculated either with poly ICLC alone (4 or 8 mg) or in combination with different doses of Adt-pIFN-α (2.5×10(9), 1×10(9), or 2.5×10(8) FFU). While different degrees of protection were achieved in all the treated animals, a dose of 8 mg of poly ICLC alone or combined with 1×10(9) FFU of Adt-pIFN-α was sufficient to sterilely protect swine when challenged 24 h later with FMDV A24. IFN-stimulated gene (ISG) expression in peripheral blood mononuclear cells at 1 day post-treatment was broader and higher in protected animals than in nonprotected animals. These data indicate that poly ICLC is a potent stimulator of IFN and ISGs in swine and at an adequate dose is sufficient to induce complete protection against FMD.

Foot-and-mouth disease virus (FMDV) leader proteinase (L(pro)) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that L(pro) is also associated with degradation of nuclear factor κB (NF-κB), a process that requires L(pro) nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the L(pro) coding sequence, SAP mutation, prevented L(pro) nuclear retention and degradation of NF-κB, resulting in in vitro attenuation. Here we report that inoculation of swine with this SAP-mutant virus does not cause clinical signs of disease, viremia, or virus shedding even when inoculated at doses 100-fold higher than those required to cause disease with wild-type (WT) virus. Remarkably, SAP-mutant virus-inoculated animals developed a strong neutralizing antibody response and were completely protected against challenge with WT FMDV as early as 2 days postinoculation and for at least 21 days postinoculation. Early protection correlated with a distinct pattern in the serum levels of proinflammatory cytokines in comparison to the levels detected in animals inoculated with WT FMDV that developed disease. In addition, animals inoculated with the FMDV SAP mutant displayed a memory T cell response that resembled infection with WT virus. Our results suggest that L(pro) plays a pivotal role in modulating several pathways of the immune response. Furthermore, manipulation of the L(pro) coding region may serve as a viable strategy to derive live attenuated strains with potential for development as effective vaccines against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is one of the most serious threats to the livestock industry. Despite the availability of a vaccine, recent outbreaks in disease-free countries have demonstrated that development of novel FMD control strategies is imperative. Here we report the identification and characterization of bovine (bo) interferon lambda 3 (IFN-λ3), a member of the type III IFN family. Expression of boIFN-λ3 using a replication-defective human adenovirus type 5 vector (Ad5-boIFN-λ3) yielded a glycosylated secreted protein with antiviral activity against FMD virus (FMDV) and vesicular stomatitis virus in bovine cell culture. Inoculation of cattle with Ad5-boIFN-λ3 induced systemic antiviral activity and up-regulation of IFN stimulated gene expression in multiple tissues susceptible to FMDV infection. Our results demonstrate that the type III IFN family is conserved in bovines and boIFN-λ3 has potential for further development as a biotherapeutic candidate to inhibit FMDV or other viruses in cattle., (Published by Elsevier Inc.)

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 pIFNβ 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.

Foot-and-mouth disease virus (FMDV) initiates translation from two in-frame AUG codons producing two forms of the leader (L) proteinase, Lab (starting at the first AUG) and Lb (starting at second AUG). In a previous study, we have demonstrated that a cDNA-derived mutant FMDV (A24-L1123) containing a 57-nucleotide transposon (tn) insertion between the two AUG initiation codons (inter-AUG region) was completely attenuated in cattle, suggesting that this region is involved in viral pathogenesis. To investigate the potential role of the Lab protein in attenuation, we have introduced two epitope tags (Flag: DYKDDDK and HA: YPYDVPDYA) or a small tetracysteine motif (tc: CCGPCC) into the pA24-L1123 infectious DNA clone. Mutant viruses with a small plaque phenotype similar to the parental A24-L1123 were recovered after transfection of constructs encoding the Flag tag and the tc motif. However, expression of the Flag- or tc-tagged Lab protein was abolished or greatly diminished in these viruses. Interestingly, the A24-L1123/Flag virus acquired an extra base in the inter-AUG region that resulted in new AUG codons in-frame with the second AUG, and produced a larger Lb protein. This N terminal extension of the Lb protein in mutant A24-L1123/Flag did not affect virus viability or L functions in cell culture., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) plays a critical role in viral pathogenesis. Molecular studies have demonstrated that L(pro) inhibits translation of host capped mRNAs and transcription of some genes involved in the innate immune response. We have used microarray technology to study the gene expression profile of bovine cells infected with wild type (WT) or leaderless FMDV. Thirty nine out of approximately 22,000 bovine genes were selectively up-regulated by 2 fold or more in leaderless versus WT virus infected cells. Most of the up-regulated genes corresponded to IFN-inducible genes, chemokines or transcription factors. Comparison of promoter sequences suggested that host factors NF-kappaB, ISGF3G and IRF1 specifically contributed to the differential expression, being NF-kappaB primarily responsible for the observed changes. Our results suggest that L(pro) plays a central role in the FMDV evasion of the innate immune response by inhibiting NF-kappaB dependent gene expression., (Published by Elsevier Inc.)

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.

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).

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, L pro, limits the host innate response by inhibiting the induction of interferon beta (IFN beta) mRNA and blocking host cell translation. A second viral proteinase, 3C pro, 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.

We have previously shown that the leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) interferes with the innate immune response by blocking the translation of interferon (IFN) protein and by reducing the immediate-early induction of beta IFN mRNA and IFN-stimulated genes. Here, we report that L(pro) regulates the activity of nuclear factor kappaB (NF-kappaB). Analysis of NF-kappaB-dependent reporter gene expression in BHK-21 cells demonstrated that infection with wild-type (WT) virus has an inhibitory effect compared to infection with a genetically engineered mutant lacking the leader coding region. The expression of endogenous NF-kappaB-dependent genes tumor necrosis factor alpha and RANTES is also reduced in WT virus-infected primary porcine cells. This inhibitory effect is neither the result of a decrease in the level of the mRNA of p65/RelA, a subunit of NF-kappaB, nor a block on the nuclear translocation of p65/RelA, but instead appears to be a consequence of the degradation of accumulated p65/RelA. Viral L(pro) is localized to the nucleus of infected cells, and there is a correlation between the translocation of L(pro) and the decrease in the amount of nuclear p65/RelA. By using a recombinant cardiovirus expressing L(pro), we demonstrate that the disappearance of p65/RelA takes place in the absence of any other FMDV product. The observation that L(pro) disrupts the integrity of NF-kappaB suggests a global mechanism by which FMDV antagonizes the cellular innate immune and inflammatory responses to viral infection.

Previously, we showed that type I interferon (alpha/beta interferon [IFN-alpha/beta]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 10(9) PFU of human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN-alpha) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-gamma) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-alpha, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 10(8) PFU of Ad5-pIFN-alpha, low- or high-dose Ad5-pIFN-gamma, or a combination of Ad5-pIFN-alpha and low- or high-dose Ad5-pIFN-gamma and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-alpha or a low dose of Ad5-pIFN-gamma developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-gamma was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-gamma or with only high-dose Ad5-pIFN-gamma were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.

We have previously shown that the leader proteinase (L(pro)) of foot-and-mouth disease virus (FMDV) blocks cap-dependent mRNA translation and that a genetically engineered FMDV lacking the leader proteinase coding region (A12-LLV2) is attenuated in cell culture and susceptible animals. The attenuated phenotype apparently is a consequence of the inability of A12-LLV2 to block the expression of type I interferon (IFN-alpha/beta) protein, resulting in IFN-induced inhibition of FMDV replication. Here we show that in addition to preventing IFN-alpha/beta protein synthesis, L(pro) reduces the level of immediate-early induction of IFN-beta mRNA and IFN-stimulated gene products such as double-stranded RNA-dependent protein kinase R (PKR), 2',5'-oligoadenylate synthetase, and Mx1 mRNAs in swine cells. Down-regulation of cellular PKR by RNA interference did not affect wild-type virus yield but resulted in a higher yield of A12-LLV2, indicating a direct role of PKR in controlling FMDV replication in the natural host. The observation that L(pro) controls the transcription of genes involved in innate immunity reveals a novel role of this protein in antagonizing the cellular response to viral infection.

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.

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]

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]

Foot-and-mouth disease virus (FMDV) causes a highly contagious disease of cloven-hoofed animals. We have previously demonstrated that a replication-defective human adenovirus 5 vector carrying the FMDV capsid coding region of serotype A24 Cruzeiro (Ad5-CI-A24-2B) protects swine and cattle against FMDV challenge by 7 days post-vaccination. However, since relatively large amounts of Ad5-CI-A24-2B are required to induce protection this strategy could be costly for livestock production. Poly ICLC is a synthetic double stranded RNA that activates multiple innate and adaptive immune pathways. In this study, we have tested for the first time, the adjuvant effect of poly ICLC in combination with Ad5-CI-A24-2B in swine. We found that the combination resulted in a reduction of the vaccine protective dose by 80-fold. Interestingly, the lowest dose of Ad5-CI-A24-2B plus 1 mg of poly ICLC protected animals against challenge even in the absence of detectable FMDV-specific neutralizing antibodies at the time of challenge. [ABSTRACT FROM AUTHOR]