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Francisco Sobrino - One of the best experts on this subject based on the ideXlab platform.

  • Innate immune sensor LGP2 is cleaved by the Leader protease of Foot-and-Mouth Disease Virus.
    PLOS Pathogens, 2018
    Co-Authors: Miguel Rodríguez Pulido, Encarnacion Martinez-salas, Maria Teresa Sánchez-aparicio, Adolfo García-sastre, Francisco Sobrino, Margarita Sáiz
    Abstract:

    The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA Viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornaVirus infection. Here, we describe the activity of the Foot-and-Mouth Disease Virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoViruses may represent a novel antagonistic mechanism for immune evasion.

  • immunogenicity and t cell recognition in swine of foot and mouth Disease Virus polymerase 3d
    Virology, 2004
    Co-Authors: Maria M Garciabriones, Esther Blanco, David Andreu, Cristina Chiva, Victoria Ley, Francisco Sobrino
    Abstract:

    Immunization of domestic pigs with a vaccinia Virus (VV) recombinant expressing Foot-and-Mouth Disease Virus (FMDV) 3D protein conferred partial protection against challenge with infectious Virus. The severity reduction of the clinical symptoms developed by the challenged animals occurred in the absence of significant levels of anti-3D circulating antibodies. This observation suggested that the partial protection observed was mediated by the induction of a 3D-specific cellular immune response. To gain information on the T cell recognition of FMDV 3D protein, we conducted in vitro proliferative assays using lymphocytes from outbred pigs experimentally infected with FMDV and 90 overlapping peptides spanning the complete 3D sequence. The use of pools of two to three peptides allowed the identification of T cell epitopes that were efficiently recognized by lymphocytes from at least four of the five animals analyzed. This recognition was heterotypic because anti-peptide responses increased upon reinfection of animals with a FMDV isolate from a different serotype. The results obtained with individual peptides confirmed the antigenicity observed with peptide pools. Detection of cytokine mRNAs by RT-PCR in lymphocytes stimulated in vitro by individual 3D peptides revealed that IFN-γ mRNA was the most consistently induced, suggesting that the activated T cells belong to the Th 1 subset. These results indicate that 3D protein contains epitopes that can be efficiently recognized by porcine T lymphocytes from different infected animals, both upon primary and secondary (heterotypic) FMDV infection. These epitopes can extend the repertoire of viral T cell epitopes to be included in subunit and synthetic FMD vaccines.

  • interspecies major histocompatibility complex restricted th cell epitope on foot and mouth Disease Virus capsid protein vp4
    Journal of Virology, 2000
    Co-Authors: Esther Blanco, Kenneth C. Mccullough, Artur Summerfield, Jude Fiorini, David Andreu, Cristina Chiva, Eva Borras, P V Barnett, Francisco Sobrino
    Abstract:

    T-cell epitopes within viral polypeptide VP4 of the capsid protein of Foot-and-Mouth Disease Virus were analyzed using 15-mer peptides and peripheral blood mononuclear cells (PBMC) from vaccinated outbred pigs. An immunodominant region between VP4 residues 16 and 35 was identified, with peptide residues 20 to 34 (VP4-0) and 21 to 35 (VP4-5) particularly immunostimulatory for PBMC from all of the vaccinated pigs. CD25 upregulation on peptide-stimulated CD4+ CD8+ cells—dominated by Th memory cells in the pig—and inhibition using anti-major histocompatibility complex class II monoclonal antibodies indicated recognition by Th lymphocytes. VP4-0 immunogenicity was retained in a tandem peptide with the VP1 residue 137 to 156 sequential B-cell epitope. This B-cell site also retained immunogenicity, but evidence is presented that specific antibody induction in vitro required both this and the T-cell site. Heterotypic recognition of the residue 20 to 35 region was also noted. Consequently, the VP4 residue 20 to 35 region is a promiscuous, immunodominant and heterotypic T-cell antigenic site for pigs that is capable of providing help for a B-cell epitope when in tandem, thus extending the possible immunogenic repertoire of peptide vaccines.

  • immunogenicity of non structural proteins of foot and mouth Disease Virus differences between infected and vaccinated swine
    Archives of Virology, 1994
    Co-Authors: Ana Rodriguez, J Dopazo, Juancarlos Saiz, Francisco Sobrino
    Abstract:

    Non-structural as well as VP1 recombinant proteins of Foot-and-Mouth Disease Virus (FMDV) produced inE. coli, have been used to study the specific antibody response of infected or vaccinated swine. An analysis of sera from infected pigs, using a direct ELISA, showed that polypeptide 3ABC (spanning non-structural proteins 3A, 3B and 3C) was the most antigenic among the recombinant proteins studied and allowed specific detection of FMDV infected swine from the second week after the infection. The sensitivity of this assay was comparable to that obtained when the whole FMDV was used as ELISA antigen. Conversely, use of polypeptide 3ABC did not allow detection of significant levels of antibodies in sera from vaccinated animals. This differential pattern of ELISA reactivities offers a promising approach for the distinction of infected from vaccinated pigs. In addition, a highly specific and sensitive method of diagnosis for FMDV replication was achieved using an immunoblotting assay which detected antibodies against the 3ABC polypeptide.

  • primer design for specific diagnosis by pcr of highly variable rna Viruses typing of foot and mouth Disease Virus
    Virology, 1992
    Co-Authors: A Rodriguez, Juancarlos Saiz, Encarnacion Martinezsalas, Joaquin Dopazo, Mercedes Davila, Francisco Sobrino
    Abstract:

    Abstract A PCR assay for the specific detection and identification of viral sequences that correlate with established serotypes of Foot-and-Mouth Disease Virus (FMDV) has been developed. A new analysis based on homology profiles among reported sequences was used for primer design. RNA replicase (3D) gene regions that showed high homology among FMDVs, and low homology to other picornaViruses, were used for PCR amplification. Specific and highly sensitive detection was achieved fro RNA of FMDV types C, A, and O, either purified or extracted from vesicular fluids of infected animals, under reaction conditions permissive for the detection of variants present in the Virus population. Similarly, serotype-specific primers were designed to amplify the carboxy-terminal end of VP1 gene of FMDV types either C, A, or O. The results of PCR amplification of 15 different FMDV RNAs using type-specific primers are in agreement with the serological typing of the corresponding Viruses and show that the primer-selection procedure developed for FMDV constitutes a reliable method of viral diagnosis.

Michael Crampton - One of the best experts on this subject based on the ideXlab platform.

  • Production of Foot-and-Mouth Disease Virus SAT2 VP1 protein
    AMB Express, 2020
    Co-Authors: Mpho Victoria Mamabolo, Jacques Theron, Francois Maree, Michael Crampton
    Abstract:

    The seven serotypes of Foot-and-Mouth Disease Virus (FMDV) differ on the surface exposed regions on the VP1, 2 and 3 proteins. Amongst the three, the VP1 protein has been produced the most for use in serotyping assays for some of the Euro-Asian serotypes. In this study the VP1 protein of the FMDV SAT2/ZIM/7/83 was expressed in Escherichia coli BL21 cells in Luria broth and EnPresso^® B media in shake flasks. Production was further developed and the VP1 protein was produced at 2.15 g L^−1 in fed-batch fermentations at 2 L scale. The protein formed insoluble inclusion bodies that were isolated, denatured and refolded. When tested in ELISA, the protein was found to be highly reactive with serum from a SAT2 vaccinated guinea pig, and not reactive to SAT1 and SAT3 antisera. These results open avenues to evaluate recombinantly expressed VP1 proteins for differentiation of the three Southern African Territories serotypes of FMDV that co-occur in Southern and East Africa. In addition, this could mitigate the need for employing Virus as reagent, or having to raise reagent antibodies.

Juan M Pacheco - One of the best experts on this subject based on the ideXlab platform.

  • infection dynamics of foot and mouth Disease Virus in cattle following intranasopharyngeal inoculation or contact exposure
    Journal of Comparative Pathology, 2016
    Co-Authors: Juan M Pacheco, Carolina Stenfeldt, Luis L Rodriguez, Jonathan Arzt
    Abstract:

    For the purpose of developing an improved experimental model for studies of Foot-and-Mouth Disease Virus (FMDV) infection in cattle, three different experimental systems based on natural or simulated natural Virus exposure were compared under standardized experimental conditions. Ante-mortem infection dynamics were characterized in cattle exposed to FMDV through a novel, simulated natural intranasopharyngeal (INP) inoculation system or through standardized and controlled systems of within- or between-species direct contact exposure (cattle-to-cattle or pig-to-cattle). All three systems were efficient in causing synchronous, generalized Foot-and-Mouth Disease in cattle exposed to one of three different strains of FMDV representing serotypes O, A and Asia1. There was more within-group variation in the timing of clinical infection following natural and simulated natural Virus exposure systems when compared with the conventionally used system of needle inoculation (intraepithelial lingual inoculation). However, the three optimized exposure systems described herein have the advantage of closely simulating field conditions by utilizing natural routes of primary infection, thereby facilitating engagement of mucosal host defence mechanisms. Overall, it is concluded that INP inoculation and standardized systems of direct contact exposure provide effective alternatives to conventional (needle) inoculation systems for studies in which it is desirable to simulate the natural biology of FMDV infection.

  • infection dynamics of foot and mouth Disease Virus in pigs using two novel simulated natural inoculation methods
    Research in Veterinary Science, 2014
    Co-Authors: Carolina Stenfeldt, Juan M Pacheco, Luis L Rodriguez, Jonathan Arzt
    Abstract:

    Abstract In order to characterize Foot-and-Mouth Disease Virus (FMDV) infection dynamics in pigs, two simulated-natural inoculation systems were developed and evaluated. Intra-oropharyngeal (IOP) and intra-nasopharyngeal (INP) inoculation both enabled precise control of dose and timing of inoculation while simulating field exposure conditions. There were substantial differences between outcomes of infections by the two routes. IOP inoculation resulted in consistent and synchronous infection, whereas INP inoculation at similar doses resulted in delayed, or completely absent infection. All pigs that developed clinical infection had detectable levels of FMDV RNA in their oropharynx directly following inoculation. Furthermore, FMDV antigens were localized to the oropharyngeal tonsils suggesting a role in early infection. The utility of IOP inoculation was further demonstrated in a vaccine-challenge experiment. Thus, the novel system of IOP inoculation described herein, offers a valid alternative to traditionally used systems for FMDV inoculation of pigs, applicable for experimental studies of FMDV pathogenesis and vaccinology.

  • foot and mouth Disease Virus virulence in cattle is co determined by viral replication dynamics and route of infection
    Virology, 2014
    Co-Authors: Jonathan Arzt, Juan M Pacheco, Ethan J Hartwig, Steven J Pauszek, George R Smoliga, Teresa De Los Santos, Meghan Tucker, Elizabeth Bishop, Luis L Rodriguez
    Abstract:

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

  • characterization of cytotoxic t lymphocyte function after foot and mouth Disease Virus infection and vaccination
    Viral Immunology, 2013
    Co-Authors: Jared R Patch, Marvin J. Grubman, Juan M Pacheco, Mary Kenney, William T Golde
    Abstract:

    The induction of neutralizing antibodies specific for Foot-and-Mouth Disease Virus (FMDV) has been the central goal of vaccination efforts against this economically important Disease of cloven-hoofed animals. Although these efforts have yielded much success, challenges remain, including little cross-serotype protection and inadequate duration of immunity. Commonly, viral infections are characterized by induction of cytotoxic T lymphocytes (CTL), yet the function of CTL in FMDV immunity is poorly defined. We developed an assay for detection of CTL specific for FMDV and reported that a modified adenoVirus-vectored FMDV vaccine could induce CTL activity. This allowed us to determine whether FMDV-specific CTL responses are induced during infection and to test further whether vaccine-induced CTL could protect against challenge with FMDV. We now show the induction of antigen-specific CTL responses after infection of swine with FMDV strain A24 Cruizero. In addition, we developed a vaccination strategy that induces FMDV-specific CTL in the absence of significant neutralizing antibody. Animals vaccinated using this protocol showed delayed clinical Disease and significantly suppressed viremia compared to control animals, suggesting a role for CTLs in the control of Virus shedding. These results provide new insights showing induction of CTL responses to FMDV following infection or vaccination, and create the potential for improving vaccine performance by targeting cellular immunity.

  • mechanisms of foot and mouth Disease Virus tropism inferred from differential tissue gene expression
    PLOS ONE, 2013
    Co-Authors: James Zhu, Juan M Pacheco, Jonathan Arzt, George R Smoliga, Michael C Puckette, Luis L Rodriguez
    Abstract:

    Foot-and-Mouth Disease Virus (FMDV) targets specific tissues for primary infection, secondary high-titer replication (e.g. foot and mouth where it causes typical vesicular lesions) and long-term persistence at some primary replication sites. Although integrin αVβ6 receptor has been identified as primary FMDV receptors in animals, their tissue distribution alone fails to explain these highly selective tropism-driven events. Thus, other molecular mechanisms must play roles in determining this tissue specificity. We hypothesized that differences in certain biological activities due to differential gene expression determine FMDV tropism and applied whole genome gene expression profiling to identify genes differentially expressed between FMDV-targeted and non-targeted tissues in terms of supporting primary infection, secondary replication including vesicular lesions, and persistence. Using statistical and bioinformatic tools to analyze the differential gene expression, we identified mechanisms that could explain FMDV tissue tropism based on its association with differential expression of integrin αVβ6 heterodimeric receptor (FMDV receptor), fibronectin (ligand of the receptor), IL-1 cytokines, death receptors and the ligands, and multiple genes in the biological pathways involved in extracellular matrix turnover and interferon signaling found in this study. Our results together with reported findings indicate that differences in (1) FMDV receptor availability and accessibility, (2) type I interferon-inducible immune response, and (3) ability to clear Virus infected cells via death receptor signaling play roles in determining FMDV tissue tropism and the additional increase of high extracellular matrix turnover induced by FMDV infection, likely via triggering the signaling of highly expressed IL-1 cytokines, play a key role in the pathogenesis of vesicular lesions.

Yongxi Dou - One of the best experts on this subject based on the ideXlab platform.

Xiaoyu Guo - One of the best experts on this subject based on the ideXlab platform.

  • foot and mouth Disease Virus non structural protein 2b negatively regulates the rlr mediated ifn β induction
    Biochemical and Biophysical Research Communications, 2018
    Co-Authors: Ting Xin, Xintao Gao, Xixi Wang, Lichun Fang, Xiukun Sui, Hongfei Zhu, Shangjin Cui, Xiaoyu Guo
    Abstract:

    Foot-and-Mouth Disease Virus (FMDV) is the causative agent of Foot-and-Mouth Disease (FMD), which is an acute and highly contagious Disease affecting pigs, cattle and other cloven-hoofed animals. Several studies have shown that FMDV has evolved multiple strategies to evade the host innate immune response, but the underlying mechanisms for immune evasion are still not fully understood. In the current research, we have demonstrated that FMDV utilizes its non-structural protein 2B to sabotage the host immune response. Over-expression of the FMDV 2B inhibited Poly(I:C)-induced or SeV-triggered up-regulation of IFN-β, IL-6 as well as ISG15. When HEK293T cells were transfected with FMDV 2B, the phosphorylation of TBK1 and IRF3 was inhibited. Co-immunoprecipitation and pull-down experiments indicated that FMDV 2B protein could interact with host RIG-I and MDA5. Moreover, FMDV 2B also inhibited the expression of the RIG-I and MDA5. Thus, FMDV 2B negatively regulates the RLR-mediated IFN-β induction by targeting RIG-I and MDA5.