DDX60 selectively reduces translation off viral type II internal ribosome entry sites.

Co-opting host cell protein synthesis is a hallmark of many virus infections. In response, certain host defense proteins limit mRNA translation globally, albeit at the cost of the host cell's own protein synthesis. Here, we describe an interferon-stimulated helicase, DDX60, that decreases translation from viral internal ribosome entry sites (IRESs). DDX60 acts selectively on type II IRESs of encephalomyocarditis virus (EMCV) and foot and mouth disease virus (FMDV), but not by other IRES types or by 5' cap. Correspondingly, DDX60 reduces EMCV and FMDV (type II IRES) replication, but not that of poliovirus or bovine enterovirus 1 (BEV-1; type I IRES). Furthermore, replacing the IRES of poliovirus with a type II IRES is sufficient for DDX60 to inhibit viral replication. Finally, DDX60 selectively modulates the amount of translating ribosomes on viral and in vitro transcribed type II IRES mRNAs, but not 5' capped mRNA. Our study identifies a novel facet in the repertoire of interferon-stimulated effector genes, the selective downregulation of translation from viral type II IRES elements.

Dissecting distinct proteolytic activities of FMDV Lpro implicates cleavage and degradation of RLR signaling proteins, not its deISGylase/DUB activity, in type I interferon suppression.

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/ß) 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/ß 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/ß 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 αVß6 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/ß 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-ß gene transcription.

Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo.

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.

Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation.

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 (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 among aphthoviruses. Neither FMDV nor ERAV Lpro interfered with phosphorylation of RNA-dependent protein kinase (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.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 (Lpro). Simultaneously, we observed that Lpro 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.

The matrix domain of the Gag protein from avian sarcoma virus contains a PI(4,5)P2-binding site that targets Gag to the cell periphery.

The Gag protein of avian sarcoma virus (ASV) lacks an N-myristoyl (myr) group, but contains structural domains similar to those of HIV-1 Gag. Similarly to HIV-1, ASV Gag accumulates on the plasma membrane (PM) before egress; however, it is unclear whether the phospholipid PI(4,5)P2 binds directly to the matrix (MA) domain of ASV Gag, as is the case for HIV-1 Gag. Moreover, the role of PI(4,5)P2 in ASV Gag localization and budding has been controversial. Here, we report that substitution of residues that define the PI(4,5)P2-binding site in the ASV MA domain (reported in an accompanying paper) interfere with Gag localization to the cell periphery and inhibit the production of virus-like particles (VLPs). We show that co-expression of Sprouty2 (Spry2) or the pleckstrin homology domain of phospholipase Cδ (PH-PLC), two proteins that bind PI(4,5)P2, affects ASV Gag trafficking to the PM and budding. Replacement of the N-terminal 32 residues of HIV-1 MA, which encode its N-terminal myr signal and its PI(4,5)P2-binding site, with the structurally equivalent N-terminal 24 residues of ASV MA created a chimera that localized at the PM and produced VLPs. In contrast, the homologous PI(4,5)P2-binding signal in ASV MA could target HIV-1 Gag to the PM when substituted, but did not support budding. Collectively, these findings reveal a basic patch in both ASV and HIV-1 Gag capable of mediating PM binding and budding for ASV but not for HIV-1 Gag. We conclude that PI(4,5)P2 is a strong determinant of ASV Gag targeting to the PM and budding.

Synonymous Deoptimization of Foot-and-Mouth Disease Virus Causes Attenuation In Vivo while Inducing a Strong Neutralizing Antibody Response.

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.

Parallel single B cell transcriptomics to elucidate pig B cell repertoire.

Pork is the most widely consumed meat on the planet, placing swine health as a critical factor for both the world economy and the food industry. Infectious diseases in pigs not only threaten these sectors but also raise zoonotic concerns, as pigs can act as "mixing vessels" for several animals and human viruses and can lead to the emergence of new viruses that are capable of infecting humans. Several efforts are ongoing to develop pig vaccines, albeit with limited success. This has been largely attributed to the complex nature of pig infections and incomplete understanding of the pig immune responses. Additionally, pig has been suggested to be a good experimental model to study viral infections (e.g., human influenza). Despite the significant importance of studying pig immunology for developing infection models, zoonosis, and the crucial need to develop better swine vaccines, there is still very limited information on the response of the swine adaptive immune system to several emerging pathogens. Particularly, very little is known about the pig B cell repertoire upon infection. Understanding the B cell repertoire is especially crucial towards designing better vaccines, predicting zoonosis and can provide insights into developing new diagnostic agents. Here, we developed methods for performing parallel single pig B cell (up to 10,000 B cells) global and immunoglobulin transcriptome sequencing. We then adapted a computational pipeline previously built for human/mouse sequences, to now analyze pig sequences. This allowed us to comprehensively map the B cell repertoire and get paired antibody sequences from pigs in a single parallel sequencing experiment. We believe that these approaches will have significant implications for swine diseases, particularly in the context of swine mediated zoonosis and swine and human vaccine development.

Prophylactic treatment with PEGylated bovine IFNλ3 effectively bridges the gap in vaccine-induced immunity against FMD in cattle.

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.

Phosphoinositides direct equine infectious anemia virus gag trafficking and release.

Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.

Characterization, Directed Evolution, and Targeting of DNA Virus-Encoded RNA Capping Enzymes Using Phenotypic Yeast Platforms.

The constant and the sudden emergence of zoonotic human and animal viruses is a significant threat to human health, the world economy, and the world food supply. This has necessitated the development of broad-spectrum therapeutic strategies to combat these emerging pathogens. Mechanisms that are essential for viral replication and propagation have been successfully targeted in the past to develop broad-spectrum therapeutics that can be readily repurposed to combat new zoonotic pathogens. Because of the importance of viral RNA capping enzymes to viral replication and pathogenesis, as well as their presence in both DNA and RNA viruses, these viral proteins have been a long-standing therapeutic target. Here, we use genome sequencing information and yeast-based platforms (YeRC0M) to identify, characterize, and target viral genome-encoded essential RNA capping enzymes from emerging strains of DNA viruses, i.e., Monkeypox virus and African Swine Fever Virus, which are a significant threat to human and domestic animal health. We first identified and biochemically characterized these viral RNA capping enzymes and their necessary protein domains. We observed significant differences in functional protein domains and organization for RNA capping enzymes from emerging DNA viruses in comparison to emerging RNA viruses. We also observed several differences in the biochemical properties of these viral RNA capping enzymes using our phenotypic yeast-based approaches (YeRC0M) as compared to the previous in vitro studies. Further, using directed evolution, we were able to identify inactivation and attenuation mutations in these essential viral RNA capping enzymes; these data could have implications on virus biocontainment as well as live attenuated vaccine development. We also developed methods that would facilitate high-throughput phenotypic screening to identify broad-spectrum inhibitors that selectively target viral RNA capping enzymes over host RNA capping enzymes. As demonstrated here, our approaches to identify, characterize, and target viral genome-encoded essential RNA capping enzymes are highly modular and can be readily adapted for targeting emerging viral pathogens as well as their variants that emerge in the future.

Exploring the Molecular Basis of Vesicular Stomatitis Virus Pathogenesis in Swine: Insights from Expression Profiling of Primary Macrophages Infected with M51R Mutant Virus.

Vesicular stomatitis virus (VSV) is an emergent virus affecting livestock in the US. Previously, using a recombinant VSV carrying the M51R mutation in the matrix protein (rNJ0612NME6-M51R), we evaluated the pathogenesis of this virus in pigs. Our results indicated that rNJ0612NME6-M51R represented an attenuated phenotype in in-vivo and in ex-vivo in pig macrophages, resembling certain clinical features observed in field VSV isolates. In order to gain more insight into the molecular basis leading to the attenuation of rNJ0612NME6-M51R in pigs, we conducted a microarray analysis to assess the gene expression profiles of primary porcine macrophages infected with rNJ0612NME6-M51R compared to its parental virus (rNJ0612NME6). Our results showed an overall higher gene expression in macrophages infected with rNJ0612NME6-M51R. Specifically, we observed that the pathways related with immune cytokine signaling and interferon (IFN)-related responses (including activation, signaling, induction, and antiviral mechanisms) were the ones comprising most of the relevant genes identified during this study. Collectively, the results presented herein highlight the relevance of type I interferon during the pathogenesis of VSV in pigs. The information generated from this study may represent a framework for future studies intended to understand the molecular bases of the pathogenesis of field strains in livestock.

Evaluation of Potential In Vitro Recombination Events in Codon Deoptimized FMDV Strains.

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.

Deoptimization of FMDV P1 Region Results in Robust Serotype-Independent Viral Attenuation.

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.

Generation of Replication Deficient Human Adenovirus 5 (Ad5) Vectored FMD Vaccines.

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.

Foot-and-Mouth Disease Virus Interserotypic Recombination in Superinfected Carrier Cattle.

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.

Mutation of FMDV Lpro H138 residue drives viral attenuation in cell culture and in vivo in swine.

The foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) is a papain like protease that cleaves the viral polyprotein and several host factors affecting host cell translation and induction of innate immunity. Introduction of Lpro mutations ablating catalytic activity is not tolerated by the virus, however, complete coding sequence deletion or introduction of targeted amino acid substitutions can render viable progeny. In proof-of-concept studies, we have previously identified and characterized FMDV Lpro mutants that are attenuated in cell culture and in animals, while retaining their capacity for inducing a strong adaptive immunity. By using molecular modeling, we have now identified a His residue (H138), that resides outside the substrate binding and catalytic domain, and is highly conserved across serotypes. Mutation of H138 renders possible FMDV variants of reduced virulence in vitro and in vivo. Kinetics studies showed that FMDV A12-LH138L mutant replicates similarly to FMDV A12-wild type (WT) virus in cells that do not offer immune selective pressure, but attenuation is observed upon infection of primary or low passage porcine epithelial cells. Western blot analysis on protein extracts from these cells, revealed that while processing of translation initiation factor eIF-4G was slightly delayed, no degradation of innate sensors or effector molecules such as NF-κB or G3BP2 was observed, and higher levels of interferon (IFN) and IFN-stimulated genes (ISGs) were induced after infection with A12-LH138L as compared to WT FMDV. Consistent with the results in porcine cells, inoculation of swine with this mutant resulted in a mild, or in some cases, no clinical disease but induction of a strong serological adaptive immune response. These results further support previous evidence that Lpro is a reliable target to derive numerous viable FMDV strains that alone or in combination could be exploited for the development of novel FMD vaccine platforms.

Activation of the inositol (1,4,5)-triphosphate calcium gate receptor is required for HIV-1 Gag release.

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca(2+) to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca(2+) provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca(2+) stores. Following activation by binding of its ligand, IP3, it releases Ca(2+) from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca(2+) signaling as a potential novel cofactor in viral particle release.

Use of Protein Pegylation to Prolong the Antiviral Effect of IFN Against FMDV.

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.

Use of IFN-Based Biotherapeutics to Harness the Host Against Foot-And-Mouth Disease.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals that severely constrains international trade of livestock and animal products. Currently, disease control measures include broad surveillance, enforcement of sanitary policy, and use of an inactivated vaccine. While use of these measures has contributed to eliminating foot-and-mouth disease virus (FMDV) from a vast area of the world, the disease remains endemic in three continents, and outbreaks occasionally appear in previously declared FMD-free zones, causing economic and social devastation. Among others, a very fast rate of viral replication and the need for 7 days to achieve vaccine-induced protection are the main limitations in controlling the disease. New fast-acting antiviral strategies targeted to boost the innate immunity of the host to block viral replication are needed. Here we review the knowledge on the multiple strategies FMDV has evolved to block the host innate immunity, with particularly focus on the past and current research toward the development of interferon (IFN)-based biotherapeutics in relevant livestock species.

Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that "synonymous codon deoptimization" could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.