5 INHIBITION OF FOOT AND MOUTH DISEASE VIRUS IN VITRO USING RNA INTERFERENCE

2009 ◽  
Vol 21 (1) ◽  
pp. 103
Author(s):  
M. Peoples ◽  
S. Sadeghieh ◽  
E. Hwang ◽  
S. Chen ◽  
G. Hannon ◽  
...  
Keyword(s):  
Cell Line ◽  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Great Promise ◽  
Starting Point ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Resistance To Infection

The use of short-hairpin RNA (shRNA) targeting viral genomes has shown great promise in human medicine and in vitro research in animal agriculture. However, this research has not been extrapolated into livestock applications. Foot and mouth disease virus (FMDV) is a world-wide disease resulting in decreased production and export limitations in countries with endemic FMDV, as well as severe economical impacts if an outbreak occurs in an FMDV-free country. The long-term goal for this project is to produce transgenic cattle that express shRNA targeting the FMDV genome resulting in resistance to infection. As a starting point, five siRNA and one non-targeting control siRNA (Null) were developed targeting different highly conserved regions of a FMDV type-A based replicon. The siRNA were transfected into BHK cells 48 h before viral RNA challenge. Eighteen hours post challenge the cells were lysed and analyzed. Three siRNA targeting the non-structural polymerase protein exhibited severe knockdown of 87, 90, and 92% when compared with the Null siRNA transfected control. The siRNA targeting the VPG3 cap protein reduced activity by 59%, and the siRNA targeting the internal ribosomal entry site had a minimal effect of 15% reduction. Based upon these results, we produced recombinant lentiviral particles designed to deliver the shRNA sequence targeting the FMDV genome and the fluorescent marker, dsRed, into a bovine fetal fibroblast cell line. This transgenic cell line expressing the most effective shRNA (based on initial siRNA screening) was used for somatic cell nuclear transfer to create bovine embryos. One hundred and sixty oocytes were enucleated, of which 149 had successful fusion resulting in 35 blastocysts after in vitro culture. Two embryos per recipient were transferred into five recipients. At Day 40 of pregnancy three of the five recipients had a fetus, but no heart beat could be detected. We are currently in the process of creating another cell line and repeating this experiment. If successful, transgenic calves will be visually and genetically analyzed for expression of dsRed and shRNA targeting FMDV. Transgenic and control animals/tissues will then be analyzed for resistance to infection with FMDV.

scholarly journals Factors Required for the Uridylylation of the Foot-and-Mouth Disease Virus 3B1, 3B2, and 3B3 Peptides by the RNA-Dependent RNA Polymerase (3Dpol) In Vitro

2005 ◽  
Vol 79 (12) ◽  
pp. 7698-7706 ◽  
Author(s):  
Arabinda Nayak ◽  
Ian G. Goodfellow ◽  
Graham J. Belsham
Keyword(s):  
Rna Polymerase ◽  
Disease Virus ◽  
Rna Synthesis ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Coding Region ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Positive Strand Rna

ABSTRACT The 5′ terminus of picornavirus genomic RNA is covalently linked to the virus-encoded peptide 3B (VPg). Foot-and-mouth disease virus (FMDV) is unique in encoding and using 3 distinct forms of this peptide. These peptides each act as primers for RNA synthesis by the virus-encoded RNA polymerase 3Dpol. To act as the primer for positive-strand RNA synthesis, the 3B peptides have to be uridylylated to form VPgpU(pU). For certain picornaviruses, it has been shown that this reaction is achieved by the 3Dpol in the presence of the 3CD precursor plus an internal RNA sequence termed a cis-acting replication element (cre). The FMDV cre has been identified previously to be within the 5′ untranslated region, whereas all other picornavirus cre structures are within the viral coding region. The requirements for the in vitro uridylylation of each of the FMDV 3B peptides has now been determined, and the role of the FMDV cre (also known as the 3B-uridylylation site, or bus) in this reaction has been analyzed. The poly(A) tail does not act as a significant template for FMDV 3B uridylylation.

scholarly journals Lithium chloride inhibits early stages of foot-and-mouth disease virus (FMDV) replication in vitro

2017 ◽  
Vol 89 (11) ◽  
pp. 2041-2046 ◽  
Author(s):  
Fu-Rong Zhao ◽  
Yin-Li Xie ◽  
Ze-Zhong Liu ◽  
Jun-Jun Shao ◽  
Shi-Fang Li ◽  
...  
Keyword(s):  
Disease Virus ◽  
Lithium Chloride ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Early Stages ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Fmdv Replication

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

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Gisselle N. Medina ◽  
Paul Azzinaro ◽  
Elizabeth Ramirez-Medina ◽  
Joseph Gutkoska ◽  
Ying Fang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Type I ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
Infected Cells ◽  
Viral Attenuation

ABSTRACT 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.

scholarly journals In vitro comparison of foot-and-mouth disease virus subtype variants causing disease in vaccinated cattle

1978 ◽  
Vol 80 (3) ◽  
pp. 451-459 ◽  
Author(s):  
E. C. Anderson ◽  
W. J. Doughty ◽  
J. Anderson ◽  
D. Baber
Keyword(s):  
Disease Virus ◽  
Vaccine Strain ◽  
Complement Fixation ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Radial Immunodiffusion ◽  
Antibody Concentration ◽  
Mouth Disease Virus ◽  
Foot And Mouth

SummaryFoot-and-mouth disease virus isolates of types O, A and SAT 2, from diseased animals in herds routinely vaccinated twice a year were compared antigenically with the vaccine strains in the complement-fixation, neutralization and radial immunodiffusion tests. It was found that strains which had readily infected vaccinated cattle had R values against the vaccine strain in the complement- fixation and radial immunodiffusion tests of 30 or less, while strains causing primary outbreaks with little spread had R values of 30–40. Threefold differences in humoral neutralizing antibody concentration between the field variant and the vaccine strain in sera from vaccinated animals were likely to be significant in terms of protection.

scholarly journals Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

2009 ◽  
Vol 83 (21) ◽  
pp. 11275-11282 ◽  
Author(s):  
Stewart Goodwin ◽  
Tobias J. Tuthill ◽  
Armando Arias ◽  
Richard A. Killington ◽  
David J. Rowlands
Keyword(s):  
Disease Virus ◽  
Receptor Binding ◽  
Virus Assembly ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Precursor Protein ◽  
Capsid Precursor ◽  
Mouth Disease Virus ◽  
Foot And Mouth

ABSTRACT The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in E scherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin αvβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.

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