¿Cómo mejoramos la participación comunitaria en salud? Análisis de las acciones propuestas tras la aplicación de una herramienta de evaluación / How to improve community engagement in health: Analysis of proposed actions following the implementation of a community engagement assessment tool

Objetivo Analizar las líneas de acción propuestas por los proyectos de promoción de la salud participantes en el proyecto de investigación EvaluAGPS, y su relación con las puntuaciones obtenidas con EvalGuía, una herramienta para evaluar la participación comunitaria basada en la evidencia. Diseño Estudio multicéntrico cuali-cuantitativo. Emplazamiento Proyectos de atención primaria o de red intersectorial de atención primaria y municipalidades de 5 comunidades autónomas en España. Participantes Personas que trabajan en 10 proyectos de promoción de la salud, seleccionados con muestreo intencional según criterios de inclusión (proyectos con un mínimo de participación comunitaria centrados en la salud comunitaria). Método Se recogieron los datos mediante cuestionarios (herramienta EvalGuía) y talleres participativos. Los datos cuantitativos se analizaron con estadística descriptiva, los datos cualitativos se analizaron utilizando el análisis de matriz. Resultados Tras pasar la herramienta EvalGuía, las puntuaciones más bajas estaban en la evaluación de resultados, conocimiento de leyes relacionadas con participación comunitaria, diversidad en el grupo motor, medidas de conciliación, recursos financieros y devolución de resultados. Las líneas de acción planteadas eran heterogéneas y no siempre coinciden con las priorizadas. Las líneas priorizadas giraban en torno a la organización del proyecto y a la comunicación. Conclusiones La herramienta EvalGuía puede ser útil para diseñar planes de acción en proyectos de promoción de la salud. La implementación de medidas en 12 meses para aumentar la diversidad del grupo motor, incorporar medidas de conciliación o mejorar la evaluación es difícil. Se requiere más tiempo para implementar este tipo de medidas. (AU)

Objective To analyse the lines of action identified in the health promotion projects participating in the EvaluA GPS research, and their relationship with the scores assigned in EvalGuia, a tool for evaluating evidence-based community participation. Design Qualitative-quantitative multicentre study. Setting Primary care or intersectoral network of primary care and municipalities in five autonomous communities in Spain. Participants Participants of 10 health promotion projects, selected with convenience sampling, following inclusion criteria (projects with a minimum of community engagement and centred on community health). Method Data were collected through questionnaires (EvalGuía tool) and participatory workshops. Quantitative data were analysed with descriptive statistics, qualitative data were analysed using matrix analysis. Results After implementing the EvalGuide tool, the lowest scores were assigned in outcome evaluation, knowledge of policies related to community participation, diversity in the core working group, inclusivity policies, financial resources and diffusion of results. The lines of action proposed were heterogeneous and did not always match with those prioritised as lower score. The prioritised lines revolved around project organisation and communication. Conclusions The EvalGuide tool can be helpful to design action plans in Health Promotion projects. The implementation of measures in 12 months to increase the diversity of the core working group, to incorporate work–life balance measures or to improve evaluation is difficult. More time is needed to implement such measures. (AU)

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.

[How to improve community engagement in health: Analysis of proposed actions following the implementation of a community engagement assessment tool]. / ¿Cómo mejoramos la participación comunitaria en salud? Análisis de las acciones propuestas tras la aplicación de una herramienta de evaluación.

OBJECTIVE: To analyse the lines of action identified in the health promotion projects participating in the EvaluA GPS research, and their relationship with the scores assigned in EvalGuia, a tool for evaluating evidence-based community participation. DESIGN: Qualitative-quantitative multicentre study. SETTING: Primary care or intersectoral network of primary care and municipalities in five autonomous communities in Spain. PARTICIPANTS: Participants of 10 health promotion projects, selected with convenience sampling, following inclusion criteria (projects with a minimum of community engagement and centred on community health). METHOD: Data were collected through questionnaires (EvalGuía tool) and participatory workshops. Quantitative data were analysed with descriptive statistics, qualitative data were analysed using matrix analysis. RESULTS: After implementing the EvalGuide tool, the lowest scores were assigned in outcome evaluation, knowledge of policies related to community participation, diversity in the core working group, inclusivity policies, financial resources and diffusion of results. The lines of action proposed were heterogeneous and did not always match with those prioritised as lower score. The prioritised lines revolved around project organisation and communication. CONCLUSIONS: The EvalGuide tool can be helpful to design action plans in Health Promotion projects. The implementation of measures in 12 months to increase the diversity of the core working group, to incorporate work-life balance measures or to improve evaluation is difficult. More time is needed to implement such measures.

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.

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.

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.

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.

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.

Treatment with Ad5-Porcine Interferon-α Attenuates Ebolavirus Disease in Pigs.

Under experimental conditions, pigs infected with Ebola Virus (EBOV) develop disease and can readily transmit the virus to non-human primates or pigs. In the event of accidental or intentional EBOV infection of domestic pigs, complex and time-consuming safe depopulation and carcass disposal are expected. Delaying or preventing transmission through a reduction in viral shedding is an absolute necessity to limit the spread of the virus. In this study, we tested whether porcine interferon-α or λ3 (porIFNα or porIFNλ3) delivered by a replication-defective human type 5 adenovirus vector (Ad5-porIFNα or Ad5-porIFNλ3) could limit EBOV replication and shedding in domestic pigs. Our results show that pigs pre-treated with Ad5-porIFNα did not develop measurable clinical signs, did not shed virus RNA, and displayed strongly reduced viral RNA load in tissues. A microarray analysis of peripheral blood mononuclear cells indicated that Ad5-porIFNα treatment led to clear upregulation in immune and inflammatory responses probably involved in protection against disease. Our results indicate that administration of Ad5-porIFNα can potentially be used to limit the spread of EBOV in pigs.

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.

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.

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.

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.

The VP1 G-H loop hypervariable epitope contributes to protective immunity against Foot and Mouth Disease Virus in swine.

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-FMDxeno, or PBS, followed by intradermal challenge with FDMV strain O1 Manisa at 21 days post-vaccination. While overall serum antibody titers were significantly higher in Ad5-FMDxeno vaccinated animals, neutralizing antibody titers were decreased in pigs that received Ad5-FMDxeno, 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-FMDxeno 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.

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 Different Tactics of Foot-and-Mouth Disease Virus to Evade Innate Immunity.

Like all pathogens, foot-and-mouth disease virus (FMDV) is recognized by the immune system inducing a heightened immune response mainly mediated by type I and type III IFNs. To overcome the strong antiviral response induced by these cytokines, FMDV has evolved many strategies exploiting each region of its small RNA genome. These include: (a) inhibition of IFN induction at the transcriptional and translational level, (b) inhibition of protein trafficking; (c) blockage of specific post-translational modifications in proteins that regulate innate immune signaling; (d) modulation of autophagy; (e) inhibition of stress granule formation; and (f) in vivo modulation of immune cell function. Here, we summarize and discuss FMDV virulence factors and the host immune footprint that characterize infection in cell culture and in the natural hosts.

Mek1 coordinates meiotic progression with DNA break repair by directly phosphorylating and inhibiting the yeast pachytene exit regulator Ndt80.

Meiotic recombination plays a critical role in sexual reproduction by creating crossovers between homologous chromosomes. These crossovers, along with sister chromatid cohesion, connect homologs to enable proper segregation at Meiosis I. Recombination is initiated by programmed double strand breaks (DSBs) at particular regions of the genome. The meiotic recombination checkpoint uses meiosis-specific modifications to the DSB-induced DNA damage response to provide time to convert these breaks into interhomolog crossovers by delaying entry into Meiosis I until the DSBs have been repaired. The meiosis-specific kinase, Mek1, is a key regulator of meiotic recombination pathway choice, as well as being required for the meiotic recombination checkpoint. The major target of this checkpoint is the meiosis-specific transcription factor, Ndt80, which is essential to express genes necessary for completion of recombination and meiotic progression. The molecular mechanism by which cells monitor meiotic DSB repair to allow entry into Meiosis I with unbroken chromosomes was unknown. Using genetic and biochemical approaches, this work demonstrates that in the presence of DSBs, activated Mek1 binds to Ndt80 and phosphorylates the transcription factor, thus inhibiting DNA binding and preventing Ndt80's function as a transcriptional activator. Repair of DSBs by recombination reduces Mek1 activity, resulting in removal of the inhibitory Mek1 phosphates. Phosphorylation of Ndt80 by the meiosis-specific kinase, Ime2, then results in fully activated Ndt80. Ndt80 upregulates transcription of its own gene, as well as target genes, resulting in prophase exit and progression through meiosis.