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Detergent-solubilized RNA polymerase from cells infected with foot-and-mouth disease virus.

The foot-and-mouth disease virus RNA polymerase complex was dissociated from cellular membranes with deoxycholate in the presence of dextran sulfate. The soluble polymerase complex was active in the cell-free synthesis of virus-specific RNA; solubilization of the complex permitted direct analysis of the cell-free reaction mixtures without recourse to RNA extraction. A major RNA-containing component found early during cell-free incubation ranged from approximately 140 to 300S. The final major products of the cell-free system were 37S virus RNA, 20S ribonuclease-resistant RNA, and a 50S component containing RNA.

Aphthovirus↗

Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids.

Foot-and-mouth disease virus (FMDV) manifests an extreme sensitivity to acid, which is thought to be important for entry of the RNA genome into the cell. We have compared the low-pH-induced disassembly in vitro of virions and natural empty capsids of three subtypes of serotype A FMDV by enzyme-linked immunosorbent assay and sucrose gradient sedimentation analysis. For all three subtypes (A22 Iraq 24/64, A10(61), and A24 Cruzeiro), the empty capsid was more stable by 0.5 pH unit on average than the corresponding virion. Unexpectedly, in the natural empty capsids used in this study, the precursor capsid protein VP0 was found largely to be cleaved into VP2 and VP4. For picornaviruses the processing of VP0 is closely associated with encapsidation of viral RNA, which is considered likely to play a catalytic role in the cleavage. Investigation of the cleavage of VP0 in natural empty capsids failed to implicate the viral RNA. However, it remains possible that these particles arise from abortive attempts to encapsidate RNA. Empty capsids expressed from a vaccinia virus recombinant showed essentially the same acid lability as natural empty capsids, despite differing considerably in the extent of VP0 processing, with the synthetic particles containing almost exclusively uncleaved VP0. These results indicate that it is the viral RNA that modulates acid lability in FMDV. In all cases the capsids dissociate at low pH directly into pentameric subunits. Comparison of the three viruses indicates that FMDV A22 Iraq is about 0.5 pH unit more sensitive to low pH than types A10(61) and A24 Cruzeiro. Sequence analysis of the three subtypes identified several differences at the interface between pentamers and highlighted a His-alpha-helix dipole interaction which spans the pentamer interface and appears likely to influence the acid lability of the virus.

Amino Acid Sequence↗

Identification of the active-site residues of the L proteinase of foot-and-mouth disease virus.

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displayed wild-type viral protein synthesis and host shut-off kinetics.

Animals↗

Cross-reactive idiotopes among anti-foot and mouth disease virus neutralizing antibodies.

Foot and mouth disease virus (FMDV) viral protein 1 is the only one of the four viral proteins (VP) that induces neutralizing antibodies as an isolated protein. A 32 amino acid (AA) residue (32dimer) of FMDV subtype A12 Lp ab VP1 (AA 137-168) was immunogenic against the A12 subtype. Three antibody populations each recognizing different epitopes on 32dimer were isolated by affinity chromatography (AFC) from the serum of a steer which had been immunized with the 32dimer. The 32dimer contains an AA sequence that is recognized by a protective paratope carried on a murine monoclonal antibody (mAb) (7SF-3.H3.1). Polyclonal anti-7SF-3 idiotype antibodies specifically inhibited the binding activity of one of these anti-32dimer antibody populations suggesting the existence of cross-reactive paratopic-related idiotopes between mAb 7SF-3 and antibodies elicited by the 32dimer. These anti-idiotypic antibodies were used in AFC to purify antibodies from the anti-32dimer serum. The purified antibody population has characteristics that resemble those of the mAb 7SF-3, i.e. its reactivity with FMDV A subtypes in ELISA, radioimmunoassay (RIA), mouse neutralization and its lack of reactivity with a mAb 7SF-3 neutralizing escape virus variant. Furthermore, these antibodies were specifically inhibited by either anti-mAb 7SF-3 idiotypic antibodies or peptides containing the mAb 7SF-3 epitope. Using the same experimental approach, mAb 7SF-3 idiotope-bearing antibodies were shown to be present in serum from bovine and swine convalescent from FMDV A12 Lp ab infection. Thus, the highly immunogenic area between residues 137 and 168 of FMDV VP1 elicited a cross-reactive neutralizing idiotope response conserved amongst several animal species.

Animals↗

A simulation model of intraherd transmission of foot and mouth disease with reference to disease spread before and after clinical diagnosis.

Intraherd transmission of foot and mouth disease virus (FMDV) was examined using a simulation model for a hypothetical 1,000-cow dairy, assuming clinical diagnosis was made when at least 1% (10 cows) or 5% (50 cows) had clinical signs of FMD, I index case cow, and transition state distributions for the latent, subclinically infectious, and clinically infectious periods of FMD calculated from published data. Estimates assumed for the number of animal-to-animal contacts (k) adequate for transmission ranged from 0.6 to 9.0 per hour (13.7-216.0 per day). A total of 40,000 iterations (5,000 for each scenario, assessing 4 adequate contact rates and 2 detection criteria) were run. The model predicted that FMD would not be diagnosed in the herd until 10.0-13.5 days after the index case cow had become infected, at which time between 65% and 97% of the cows (646-967 cows) to nearly 100% (978-996 cows) would already have become infected with the virus, if the number of cows showing clinical signs of FMD at the time of diagnosis were 10 or 50, respectively. At the time of diagnosis, the simulated number of infectious cattle varied substantially from 82-472 to 476-537 cows, depending on adequate contact rate and whether the diagnosis was made when 10 or 50 animals were showing clinical signs, respectively. The simulated number of infectious cows increased rapidly during the first few days after diagnosis. In the scenario where at least 10 cows showing clinical signs was necessary before a clinical diagnosis was made, each day after diagnosis, the number of infectious animals increased by nearly 100 to more than 200 cases per day up to day 5, assuming 0.57-9.0 animal-to-animal contacts per hour, respectively. Results obtained when it was assumed that at least 50 clinical cases were present at the time of diagnosis showed smaller relative increases because nearly one-half of the herd was projected to be infected at the time of diagnosis. From these results, it is clear that once an individual in a herd becomes infected with FMDV, herd infectivity is not static, rather it accelerates as would be expected as long as there are sufficient susceptible animals to sustain the increasing transmission rate, after which time the rate at which new infections occurs will diminish. Results indicate that biosecurity strategies aimed at minimizing both intraherd and interherd contact will be critical in minimizing the spread of FMD before the initial diagnosis is made. In addition, simulations suggest that very early clinical diagnosis of FMD and effective isolation or depopulation and disposal will be critical in limiting the number of infectious animals capable of transmitting the virus to other herds and thus in timely control of an epidemic. Early diagnosis will rely on early virus detection from animals in the preclinical phase of infection, rather than waiting for clinical signs to manifest in sufficient numbers to be noticed and to warrant investigation.

Animals↗

Acyclovir in the treatment of hand-foot-and-mouth disease.

Twelve children ages 1 to 5 years and one adult with hand-foot-and-mouth disease were treated with oral acyclovir within one to two days of onset of the rash. Symptomatic relief, defervescence, and significant involution of lesions were seen within twenty-four hours of initiating therapy. Acyclovir was continued for five days, by which time palmar, plantar, and oral lesions were virtually gone. Acyclovir is a molecule tailored to inactivate the thymidine kinase of the herpesvirus. Since the Coxsackie A16 virus causing hand-foot-and-mouth disease lacks this enzyme, the beneficial therapeutic effect must be explained on other grounds, possibly due to enhancement of the antiviral effect of the patient's own interferon.

Acyclovir↗

Interferon inducers and foot-and-mouth disease vaccines: influence of two synthetic polynucleotides on antibody response and immunity in guinea pigs and swine.

Polyriboadenylic-polybouridylic acid enhanced the immunological response of guinea pigs to aqueous foot-and-mouth disease virus vaccine. Polyriboninosinic-polyribocytidylic acid enhanced the early antibody production of swine to oil emulsified foot-and-mouth disease virus vaccine. Polyriboninosinic-polyribocytidylic acid alone did not stimulate resistance to foot-and-mouth disease in swine.

Animals↗

Use of pre-coated immunoplates and freeze-dried reagents for the diagnosis of foot-and-mouth disease and swine vesicular disease by enzyme-linked immunosorbent assay (ELISA).

An indirect sandwich ELISA is used by the World Reference Laboratory for Foot-and-Mouth Disease for the diagnosis of foot-and-mouth disease virus and swine vesicular disease virus. The potential for supplying ELISA 'kits' for diagnosis to other laboratories has been assessed by evaluating the reactivity of (a) immunoplates pre-coated with rabbit antisera to FMDV and SVDV and (b) freeze-dried diluted reference antisera. Immunoplates pre-coated using a sodium carbonate/hydrogen carbonate buffer retained 100% sensitivity at temperatures of 4 degrees C and -20 degrees C over the experimental storage period of 140 days but elevated storage temperatures, 18-24 degrees C and 37 degrees C, produced declining reactivity. There was a marked improvement in retention of reactivity upon storage at 37 degrees C when employing an alternative coating buffer, ammonium hydrogen carbonate. The reactivity of the rabbit antisera diluted in sodium carbonate/hydrogen carbonate solution and freeze-dried was high, as was the freeze-dried guinea pig antisera which had been diluted in each of the test solutions investigated. ELISA 'kits' for diagnosis, therefore, could be supplied using pre-coated immunoplates, with freeze-dried antiserum reagents or a combination of the two.

Animals↗

A point pattern model of the spread of foot-and-mouth disease.

The spatial spread of foot-and-mouth disease (FMD) is influenced by several sources of spatial heterogeneity: heterogeneity of the exposure to the virus, heterogeneity of the animal density and heterogeneity of the networks formed by the contacts between farms. A discrete space model assuming that farms can be reduced to points is proposed to handle these different factors. The farm-to-farm process of transmission of the infection is studied using point-pattern methodology. Farm management, commercial exchanges, possible airborne transmission, etc. cannot be explicitly taken into account because of lack of data. These latter factors are introduced via surrogate variables such as herd size and distance between farms. The model is built on the calculation of an infectious potential for each farm. This method has been applied to the study of the 1967-1968 FMD epidemic in UK and allowed us to evaluate the spatial variation of the probability of infection during this epidemic. Maximum likelihood estimation has been conducted conditional on the absence of data concerning the farms which were not infected during the epidemic. Model parameters have then been tested using an approximated conditional-likelihood ratio test. In this case study, results and validation are limited by the lack of data, but this model can easily be extended to include other information such as the effect of wind direction and velocity on airborne spread of the virus or the complex interactions between the locations of farms and the herd size. It can also be applied to other diseases where point approximation is convenient. In the context of an increase of animal density in some areas, the model explicitly incorporates the density and known epidemiological characteristics (e.g. incubation period) in the calculation of the probability of FMD infection. Control measures such as vaccination or slaughter can be simply introduced, respectively, as a reduction of the susceptible population or as a reduction of the source of infection.

Animal Husbandry↗

The structure of foot-and-mouth disease virus.

Structural studies of foot-and-mouth disease virus (FMDV) have largely focused on the mature viral particle, providing atomic resolution images of the spherical protein capsid for a number of sero- and sub-types, structures of the highly immunogenic surface loop, Fab and GAG receptor complexes. Additionally, structures are available for a few non-structural proteins. The chapter reviews our current structural knowledge and its impact on our understanding of the virus life cycle proceeding from the mature virus through immune evasion/inactivation, cell-receptor binding and replication and alludes to future structural targets.

Capsid Proteins↗

Modeled detection time for surveillance for foot-and-mouth disease virus in bulk tank milk.

OBJECTIVE: To estimate when foot-and-mouth disease virus (FMDV) would first be detected in bulk tank milk of dairies after exposure to FMDV. SAMPLE POPULATION: Hypothetical dairy herds milking 100, 500, or 1,000 cows. PROCEDURES: For each day after herd exposure to FMDV, infection, milk yield, and virolactia were simulated for individual cows with low and high rates of intraherd transmission to estimate when a PCR assay would detect virus in bulk tank milk. Detection limits were based on assumptions for the number of virus genomes per milliliter of milk and for analytical sensitivity of a PCR assay. RESULTS: A mean of 10% of the cows was predicted to have FMD lesions from 7 to 8 days and from 13.5 to 15 days after herd exposure for herds with high and low intraherd transmission rates, respectively. Herd bulk milk volume decreased by 10% by 8.5 to 9.5 days and by 15 to 16.5 days after herd exposure for herds with high and low transmission rates, respectively. Mean times by which FMDV would be first detected in bulk milk were 2.5 days and 6.5 to 8 days after herd exposure, which were extended for 10 to 11 days and 17 to 18 days for herds with high and low transmission rates, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: PCR screening of bulk milk for FMDV would likely detect FMDV in dairy herds several days sooner than might be expected for owner reporting of clinical signs and thus should be worthy of consideration for regional, national, or global FMD surveillance.

Animals↗

Foot-and-mouth disease virus (FMDV) causes an acute disease that can be lethal for adult laboratory mice.

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an acute vesicular disease of cloven-hoofed animals. This virus continues to be threat to livestock worldwide with outbreaks causing severe economic losses. However, very little is known about FMDV pathogenesis, partially due to the inconveniences of working with cattle and swine, the main natural hosts of the virus. Here we demonstrate that C57BL/6 and BALB/C adult mice are highly susceptible to FMDV infection when the virus is administered subcutaneously or intraperitoneally. The first clinical signs are ruffled fur, apathy, humped posture, and wasting, which are followed by neurological signs such as hind-limb paralysis. Within 2-3 days of disease onset, the animals die. Virus is found in all major organs, indicating a systemic infection. Mice developed microvesicles near the basal layer of the epithelium, event that precedes the vesiculation characteristics of FMD. In addition, a lymphoid depletion in spleen and thymus and severe lymphopenia is observed in the infected mice. When these mice were immunized with conventional inactivated FMDV vaccine, they were protected (100% of vaccinated animals) against challenge with a lethal dose of FMDV. The data indicate that this mouse model may facilitate the study of FMDV pathogenesis, and the development of new effective vaccines for FMD.

Acute Disease↗

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing.

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.

Animals↗

Managing an animal health emergency in Taipei China: foot and mouth disease.

Taipei China had been free from foot and mouth disease (FMD) over 68 years before the disease occurred in March 1997. The first suspected case was recorded on a pig farm in the Hsinchu Prefecture on 14 March 1997. Based on clinical signs, gross histopathological findings, and results of enzyme-linked immunosorbent assays and reverse-transcriptase polymerase chain reaction tests, diagnosis of FMD was confirmed by the Taiwan Animal Health Research Institute on 19 March 1997 and was reconfirmed by the FMD World Reference Laboratory in Pirbright (United Kingdom), on 25 March 1997. By the end of July 1997, 6,147 pig farms (about a quarter of the pig farms in Taipei China), were affected. The disease was well under control within two months by means of stamping-out and blanket vaccination. The Government purchased 21 million doses of inactivated oil-adjuvant FMD vaccine, which allowed for two injections per pig and one injection of other cloven-hoofed animals. Before the vaccine was used, the stamping-out policy was implemented, ensuring that all pigs in the affected farms were destroyed. After blanket vaccination, a partial stamping-out policy was adopted, i.e. only pigs showing clinical signs were destroyed.

Animals↗

[Use of the radial immunodiffusion test in the study of foot-and-mouth disease viruses].

The radial immunodiffusion test (RIT) was used in the study of the quantitative interrelations between three subtypes of type A of the foot-and-mouth disease virus--A5, A10, and A22. A reverse proportional correlation was demonstrated between the size of the precipitation circles and the antibody concentration in the homologous sera, and a direct proportional correlation between the diameter of the circles and the amount of the antigen used. It was shown that one and the same antigen produced precipitation circles of a different size with several series of homologous hyperimmune serum, depending on the content of antibodies in these sera. It was found in experiments with early sera obtained on the 7th day of a foot-and-mouth disease infection in guinea pigs as well as in investigations with antiserum 140 S of the foot-and-mouth disease virus A5 that the early and 140 S sera used in RIT had higher specificity as against the hyperimmune sera. Stated is the necessity of checking the specificity of the early sera, which often reveals antibodies of the 7 S class.

Animals↗

Protective immune response against foot-and-mouth disease.

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of the Aphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Th-lymphocyte epitopes (4, 5, 7, 8), although a Th-lymphocyte-independent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19).

Animals↗

Specificity of the VP1 GH loop of Foot-and-Mouth Disease virus for alphav integrins.

Foot-and-mouth disease virus (FMDV) can use a number of integrins as receptors to initiate infection. Attachment to the integrin is mediated by a highly conserved arginine-glycine-aspartic acid (RGD) tripeptide located on the GH loop of VP1. Other residues of this loop are also conserved and may contribute to integrin binding. In this study we have used a 17-mer peptide, whose sequence corresponds to the GH loop of VP1 of type O FMDV, as a competitor of integrin-mediated virus binding and infection. Alanine substitution through this peptide identified the leucines at the first and fourth positions following RGD (RGD+1 and RGD+4 sites) as key for inhibition of virus binding and infection mediated by alphavbeta6 or alphavbeta8 but not for inhibition of virus binding to alphavbeta3. We also show that FMDV peptides containing either methionine or arginine at the RGD+1 site, which reflects the natural sequence variation seen across the FMDV serotypes, are effective inhibitors for alphavbeta6. In contrast, although RGDM-containing peptides were effective for alphavbeta8, RGDR-containing peptides were not. These observations were confirmed by showing that a virus containing an RGDR motif uses alphavbeta8 less efficiently than alphavbeta6 as a receptor for infection. Finally, evidence is presented that shows alphavbeta3 to be a poor receptor for infection by type O FMDV. Taken together, our data suggest that the integrin binding loop of FMDV has most likely evolved for binding to alphavbeta6 with a higher affinity than to alphavbeta3 and alphavbeta8.

Animals↗