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At least 19 recordsLinked to original sources
PubMed1991

Expression of the aphthovirus RNA polymerase gene in Escherichia coli and its use together with other bioengineered nonstructural antigens in detection of late persistent infections.

A plasmid has been constructed containing the DNA sequences that direct the expression of the aphthovirus RNA-dependent RNA polymerase (virus infection-associated antigen, VIAA) in its native form. The aphthovirus polypeptide was designed to contain only a single additional amino acid, the N-terminal methionine. The recombinant protein has been purified and used in enzyme-linked immunoelectrotransfer blots to detect aphthovirus-specific antibodies in the sera of persistently infected animals. Furthermore, studies were carried out to test the hypothesis that antibodies against other nonstructural antigens appear in the sera of these animals. It was established that antibodies against polypeptides 3A and 3B can serve as complementary markers for late aphthovirus-carrier state detection. The considerable potential of this approach to detect aphthovirus-specific antibodies, when the isolation of infectious virus is not possible, was demonstrated. Negative results were obtained in animals from virus-free areas and in vaccinated cattle. This assay has the added advantage that no infectious or noninfectious virus is involved during antigen production.

Animals↗
PubMed1996

Identification of an essential region for internal initiation of translation in the aphthovirus internal ribosome entry site and implications for viral evolution.

Translation of aphthovirus RNA is initiated at an internal ribosome entry site (IRES) element, preceding the first functional AUG initiation codon. The effect of mutations at the base of domain 3 of the aphthovirus IRES on translation activity has been analyzed by site-directed mutagenesis and expression of bicistronic RNAs in transfected cells. The results have shown that the enhanced IRES activity associated with a single pyrimidine transition fixed in a persistent aphthovirus variant (E. Martínez-Salas, J. C. Sáiz, M. Dávila, G. J. Belsham, and E. Domingo, J. Virol. 67:3748-3755, 1993) is base specific. Mutations predicted to destabilize the base of domain 3 were detrimental to IRES function, but subsequent restoration of the RNA structure gave rise to fully competent IRES. In contrast, single or multiple mutations that did not affect predicted helical structures modified the relative efficiency of translation by at most 10-fold, suggesting that primary sequence also plays a role in IRES activity. A correlation between the energy of stabilization of the IRES structure and the efficiency of translation has been noted. None of the 15 mutations studied reached a level of initiation of translation comparable to that of the IRES from the persistent variant. The results indicate a critical participation of the base of domain 3 in the activity of the aphthovirus IRES, with a strong effect of secondary or higher-order structures and minor effects of primary structure.

Animals↗
PubMed1993

Diagnosis of persistent aphthovirus infection and its differentiation from vaccination response in cattle by use of enzyme-linked immunoelectrotransfer blot analysis with bioengineered nonstructural viral antigens.

A highly sensitive enzyme-linked immunoelectrotransfer blot (EITB) assay, capable of detecting aphthovirus-specific antibodies to replicating virus in sera from cattle with persistent infection, was developed. The assay uses a set of purified recombinant DNA-derived nonstructural viral antigens as serologic probes in lieu of the traditionally used virus infection-associated antigen(s) partially purified from baby hamster kidney-infected cells. Sera from cattle with experimentally induced aphthovirus infection were analyzed sequentially by EITB at various postinoculation days, and the results were compared with those obtained by currently used techniques. It was established that, in all cases, EITB results remained positive at late stages of infection. At these times, results of virus infection-associated antigen-antibody determinations were negative by use of the conventional immunodiffusion in agarose gel test, and virus was recovered only occasionally from esophageal-pharyngeal fluid. Specificity of the EITB test was indicated by negative results for sera from cattle in aphthovirus-free areas, including samples from cattle infected with a variety of bovine viruses. Moreover, the test eliminated a substantial number of false-positive results (on the basis of the immunodiffusion in agarose gel assay) caused by reactivity of sera from vaccinated cattle. Use of additional nonstructural viral antigens, other than RNA polymerase, is proposed to differentiate between seropositivity resulting from vaccination or infection. This procedure may be considered to have potential applications as a sensitive, safe, rapid, and economic field test for specific diagnosis of persistent aphthovirus infection in affected animals.

Animals↗
PubMed1999

Involvement of the aphthovirus RNA region located between the two functional AUGs in start codon selection.

Initiation of translation in picornavirus RNAs occurs internally, mediated by an element termed internal ribosome entry site (IRES). In the aphthovirus RNA, the IRES element directs translation initiation at two in-frame AUGs separated by 84 nucleotides. We have found that bicistronic constructs that contained the IRES element followed by the fragment including the aphthovirus start codons in front of the second gene mimicked the translation initiation pattern of viral RNA observed in infected cells. In those constructs, the frequency of initiation at the first AUG was increased by a sequence context that resembled the favorable consensus for cap-dependent translation, although initiation at the second site was always preferred. In addition, we have found that initiation at the second start codon was not diminished under conditions in which the first initiation codon was blocked by antisense oligonucleotide interference. Interestingly, mutations that positioned the second AUG out-of-frame with the first AUG did not interfere with the frequency of initiation at the second one. On the contrary, IRES-dependent translation initiation in bicistronic constructs lacking the sequences present between functional AUGs in the viral RNA was sensitive to the presence of out-of-frame initiator codons and hairpins in the spacer region. This remarkable difference in start codon recognition was due to the nucleotide composition of the RNA that separated the IRES from the initiator codon. Thus our results indicate that the region located in the aphthovirus RNA between functional AUGs is involved in start codon recognition, strongly favoring selection of the second start AUG as the main initiator codon.

Animals↗
PubMed1983

Location of an immunizing determinant within polypeptide VP1 of type O aphthovirus.

VP1 is the only structural polypeptide of aphthovirus able to stimulate the production of neutralizing antibody. The region of VP1 responsible for this activity was located by testing various proteolytic fragments of VP1 for their ability to compete for virus-specific antibodies in serum raised against the intact polypeptide. No antigenic activity could be detected in VP1 fragments isolated from trypsin-treated virus. Controlled digestion revealed that trypsin cleaved VP1 in four places in a preferred order, whereas chymotrypsin cut at a maximum of two sites. The initial cuts by the two proteases were made very close to each other, and in each case resulted in a greatly reduced affinity of the VP1 fragments for virus-specific antibodies in anti-VP1 serum. In contrast, aphthovirus was resistant to Staphylococcus aureus V8 protease, and treatment of isolated VP1 with this protease generated a peptide of mol. wt. 8500 which competed efficiently with virus for antiserum to VP1 and for an absorbed antiviral serum specific for trypsin-sensitive sites. The results indicate that these antisera interact specifically with aphthovirus at a single antigenic determinant located on VP1 approximately two-thirds of the way along the polypeptide sequence.

Amino Acid Sequence↗
PubMed1997

Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation.

A comparison of picornavirus internal ribosome entry site (IRES) secondary structures revealed the existence of conserved motifs located on loops. We have carried out a mutational analysis to test their requirement for IRES-driven translation. The GUAA sequence, located in the aphthovirus 3A loop, did not tolerate substitutions that disrupt the GNRA motif. Interestingly, this motif was found at similar positions in all picornavirus IRESs, suggesting that it may form part of a tertiary-structure element. The RAAA tetranucleotide located in the 3B loop was conserved only in cardiovirus and aphthovirus. A mutational analysis of the RAAA motif revealed that activities of 3B loop mutants correlated with both the presence of a sequence close to CAAA at the new 3B loop and the absence of reorganization of the 3B and 3C stem-loops. In support of this conclusion, insertion of a large number of nucleotides close to the 3B loop, which was predicted to reorganize the 3B-3C stem-loop structure, led to defective IRES elements. We conclude that the aphthovirus IRES loops located at the most distal part of domain 3, which carries GNRA and RAAA motifs, are essential for IRES function.

Aphthovirus↗
PubMed1984

[Synthesis of 10S particles in cells infected with aphthovirus].

In the present study, evidence is presented for the existence of a morphogenetic intermediary that may be a precursor of the procapsids in the assembling process. BHK21 clone 13S cells were infected with Aphthovirus A24 (Cruzeiro strain), and pulse-chase experiments were carried out using 3H-leucine. Cytoplasmic extracts were then prepared at appropriate times, and analyzed by sucrose-gradient ultracentrifugation. After preliminary assays (Fig. 1), working conditions were standardized so as to obtain maximal recovery of the morphogenetic intermediary, as well as consistency of results. Only in the presence of DOC-Brij58 and Mg++ could a 10S sedimentation coefficient peak be seen (Fig. 1 a). A heterogeneous zone, with 4,5-5S as sedimentation coefficient, was also observed. The degree of labeling in the region 4,5-5S compared with that in the 10S portion, depends on the time within the infectious cycle when cells were pulse-labeled. Maximal levels for the ratio 10S/4,5-5S are reached when pulse-labeling takes place at the time when the amount of RNA viral synthesis reaches 80% of its total value (Fig. 2). Similar experiments, performed with third passage bovine fetal kidney cells, were confirmatory of the presence of a structure sedimenting at 10S, as well as of a heterogeneous zone of 4,5-5S (Fig. 3). It would appear that assembling of Aphthoviruses is accomplished through an intermediary unit which differs from that found for other Picornaviruses, the latter being the result of the union of 12 pentamers. The capsid of Aphthoviruses, also composed of 60 identical sub-units, would instead derive from the joining of 20 trimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

RNA fingerprinting of South American prototype aphthovirus strains.

Aphthovirus strains used in South America for vaccine production or as reference for diagnostic purposes were analysed by RNA fingerprinting (RNase T1 maps, one- and two-dimensional gels). The results obtained constitute the basis for a data bank containing available information about the genome structure of strains of aphthovirus prevalent in this continent and can be used as an adjunct to serological and immunological information. These data are currently being used in South American countries to assess the genetic stability of strains during vaccine production; to establish possible vaccine origin of field outbreaks and to monitor the origin, behaviour and fate of new strains in the field.

Animals↗
PubMed1984

[Humoral immune response in cattle vaccinated against aphthovirus. Study of the kinetics of protective and neutralizing antibodies].

The sera of three groups (I, II and III) of cattle vaccinated every three months with trivalent hydroxysaponinated commercial vaccine against aphthovirus were studied. The only difference between groups I and II was that the former received a revaccination on day 17 after the initial immunization. Groups I and II included sera from animals three months old born from vaccinated mothers. Group III consisted of the sera of adult animals (the mothers of animals in groups I and II). The animals from the three groups were bled monthly during one year. The studies were performed with pooled sera from each group. The presence of protective and neutralizing antibodies was investigated in the gammaglobulin fractions which were then separated into subclasses, by chromatography on DE-cellulose columns, in order to study their biological activity. The immunization of cattle 3 months old with commercial vaccine against aphthovirus resulted in weak primary humoral response; neutralizing antibodies could not be detected. When the animals were restimulated three weeks after the first immunization, neutralizing antibodies appeared although the response did not persist. Nevertheless, five months after the experiment was started both groups I and II showed neutralizing antibodies. (Fig. 1, 2, 3). Persistent immunity to the three virus subtypes was acquired by animals of groups I and II but not before nine months. The kinetics of protective antibodies was similar to that of neutralizing antibodies, but with higher titers. Some bleedings that did not show neutralizing activity, did show significant protective activity (Figs. 4, 5). The investigation of the neutralizing activity of the gammaglobulin subclasses obtained by chromatography revealed that there was not one single subclass responsible for this activity, but that several subclasses were involved. The gammaglobulin subclasses were analyzed by immunoelectrophoresis; proteins with alpha 2 mobility appeared, coincident with early bleedings of high neutralizing titers, although these proteins did not present neutralizing activity (Tables 1, 2). The protective and neutralizing activity was not correlated with the protein concentration of the fractions so that the increase observed may be due to a qualitative change in the antibodies.

Animals↗
PubMed1988

Behavior of intertypic recombinants between virulent and attenuated aphthovirus strains in tissue culture and cattle.

Two aphthovirus intertypic recombinants between the virulent strain A Venceslau and guanidine-resistant attenuated mutants of either strain C3 Resende or O1 Campos were obtained in an attempt to establish the region(s) of the viral genome responsible for attenuation in cattle. Recombinants that inherited the 3' half of the genome from either attenuated parent and the 5' half from the virulent strain were selected and analyzed with respect to their ability to grow in cells of bovine origin and for their virulence in cattle. The results obtained support our previous conclusion, derived from studies with homotypic recombinants between attenuated aphthovirus type O1 and its original virulent strain, that the host range restriction phenotype for fetal bovine kidney cells of the attenuated strain is inherited from the 3' half of the genome. For the intertypic recombinants, however, this restriction is enhanced, presumably by the presence of a heterologous 5' half of the genomic region. In addition, we demonstrate that the results in vitro correlate with those of virulence tests in cattle.

Animals↗
PubMed1982

Competition for cellular receptor sites among selected aphthoviruses.

The competition between different types of aphthoviruses (foot-and-mouth disease virus [FMDV]) for receptor site utilization was determined. The Southern African Territories (SAT) types of FMDV absorbed poorly to BHK-21 cells as measured by a radioactivity binding assay but grew to relatively high titers on these cells. On BK cells, however, all three SAT types bound well and competed with each other for receptor sites. In addition, unlabeled FMDV types A12 and O1B were able to completely inhibit the binding of 3H-uridine labeled SAT types. Unlabeled SAT, however, was only slightly able to inhibit the adsorption of labeled A12 and moderately inhibit the binding of labeled O1B. Saturation binding studies with homologous virus showed that BK cells contain at least 100-fold more receptor sites for types A12 and O1B than for the SAT types. Competitive binding analysis between type A12 FMDV and poliovirus and encephalomyocarditis virus revealed that these three viruses all used different receptor sites. Thus, different FMDV serotypes appear to utilize both common and unique receptor sites which are different from those of at least two other picornaviruses.

Animals↗
PubMed1995

Molecular evolution of aphthoviruses.

Aphthoviruses are an important group of animal pathogens. A combination of genetic and structural studies has revealed one of the main principles governing their evolution: severe limitations to variation imposed by functional and structural constraints, in conjunction with high mutation and recombination rates operating during genome replication. Evolution occurs by positive selection and random drift acting on complex quasispecies distributions. The mutant composition of a quasi-species (or mutant spectrum) is largely dictated by tolerance to nucleotide and amino acid substitutions in viral RNAs and proteins, which must remain functionally competent. We review recent evidence to support this proposal, and we suggest that similar concepts may apply to other RNA viruses as well.

Adaptation, Physiological↗
PubMed1986

Cytoskeletal association of an aphthovirus-induced polypeptide derived from the P3ABC region of the viral polyprotein.

Monolayers of BHK cells infected with aphthovirus (FMDV) were labeled for short times with [35S]methionine at 2 hr p.i. and fractionated by detergent treatment and low speed centrifugation. Polyacrylamide gel analysis showed an asymmetric distribution of the FMDV-induced polypeptides among the three different subcellular fractions obtained. Polypeptide P88-1, the viral capsid protein precursor, is mainly found in the soluble cytoplasmic extract while polypeptides P100-3, P52-2AC, P34-2C, and P14-2A are the major viral components of a detergent soluble extract of the crude nuclear pellet. Analysis of the detergent resistant fraction (DRF), which is mainly composed of cell nuclear chromatin and insoluble cytoskeletal elements, shows a clear enrichment in an incompletely characterized polypeptide which is tentatively designated P54. Variable amounts of polypeptides P100-3 and those of the P72 complex are also detected in this fraction. The preferential location of P54 in an equivalent subcellular fraction obtained by mild detergent treatment of infected monolayers in situ, and also in a high-salt resistant subfraction of the DRF, strongly suggests a close association of this polypeptide with vimentin-actin containing components of the cell. Polypeptide P54 is immunoprecipitated by viral specific antiserum from convalescent guinea pigs but not by serum against FMDV capsid proteins, indicating that it does not share common antigenic determinants with polypeptides processed from the viral capsid precursors. On the other hand, protease V8 mapping of polypeptides P100-3, P54, P88-1, and VP1-3 shows that P54 derived from the 3' end coding region of the viral genome. Further analysis by limited protease digestion also demonstrates that P54 has partial overlap with P72-3CD while it does not share any common peptide with P56a-3D, indicating that P54 contains the sequences coded in the 3ABC region of the FMDV RNA. This assumption is reinforced by the basic behavior shown by P54 in two-dimensional gels. The results support the hypothesis of a close intracellular interaction of a short-lived polypeptide, containing the viral protease and VPg sequences, with the host cytoskeleton, during infection of BHK cells with FMDV.

Animals↗
PubMed1987

Biochemical characterization of an aphthovirus type C3 strain Resende attenuated for cattle by serial passages in chicken embryos.

We have compared several aspects of an aphthovirus strain attenuated for cattle (C3R-O/E) with the original strain (C3Res) from which it was derived after serial passages in chicken embryos. Biochemical differences detected by protein analysis in regular polyacrylamide gels (SDS-PAGE) and on electrofocusing gels (NEPHGE) suggest the presence of mutations throughout the genome. Changes were located in coat proteins VP1 and VP3 and in the polymerase precursor P100 (P3/ABCD). No other differences were found at the protein level by means of the techniques used. Polypeptide P100 of the attenuated strain showed a faster electrophoretic mobility in SDS-PAGE with respect to that of the wild-type strain, and the change seems to be located on its amino terminus half. Several functional differences were also found between the two viruses. Both strains grew equally well in BHK cells reaching roughly similar titers in plaque assays. However, the wild-type strain maintained its titer in cells of bovine origin (BK), whereas the titer of C3R-O/E strain decreased approximately one log in this cell system; moreover, plaques elicited by the attenuated strain were much smaller than the ones produced by C3Res. A diminution in the rate of RNA synthesis induced by C3R-O/E in BK cells compared with that of the wild-type strain was also detected; this trait was not observed in BHK cells. A delay in the kinetics of RNA synthesis was also detected in this strain. The virus yield of attenuated strain in BK cells was four times lower than in BHK cells.

Animals↗
PubMed1988

Heterogeneity of the polyribocytidilic acid tract in aphthovirus: changes in the size of the poly(C) of viruses recovered from persistently infected cattle.

A sample of aphthovirus type C3 strain Resende carrying two polyribocytidilic acid [poly(C)] tracts was cloned in tissue culture. One clone with a poly(C)-rich tract of about 145 nucleotides long (clone 3B) and another with a poly(C)-rich tract of about 230 nucleotides long (clone 12) and a mixture of both were injected intralingually into three steers. Samples from all three animals were recovered during the acute phase of the disease, from the blood and from the feet, and at various days after inoculation from the oesophageal-pharyngeal (OP) fluids. Analysis of the viral RNAs of the positive samples by means of RNase T1 maps on one- and two-dimensional gels showed (1) changes in the electrophoretic mobility of the poly(C)-rich tracts of viruses recovered from the OP fluids at various times after infection; (2) selection of virus populations with poly(C)-rich tracts of increased size; (3) later on, changes in the patterns of oligonucleotides of persistent viruses. These variations may lead to the production of new strains with altered biological properties that may contribute to the maintenance and spread of these viruses in the field.

Animals↗
PubMed1980

The structural polypeptides of aphthovirus are phosphoproteins.

Analysis of aphthovirus A12, strain 119ab, grown in the presence of inorganic 32P revealed that two of the major viral polypeptides, VP4 and trypsin-sensitive protein VP3, were highly phosphorylated. The other major polypeptides, VP1 and VP2, were also phosphorylated but to a much lesser extent. Polypeptides VP0 and P56, of which there are approximately one of two copies per aphthovirion, were also labeled with 32P. Phosphoserine and phosphothreonine appeared to be the amino acids labeled with 32P.

Aphthovirus↗
PubMed1996

Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses.

Equine rhinoviruses (ERVs) are picornaviruses which cause a mild respiratory infection in horses. The illness resembles the common cold brought about by rhinoviruses in humans; however, the presence of a viraemia during ERV-1 infection, the occurrence of persistent infections and the physical properties are all more reminiscent of foot-and-mouth disease virus (FMDV). cDNA cloning and sequencing of the genomes of ERV-1 and ERV-2 between the poly(C) and poly(A) tracts showed that the serotypes are heterogeneous. Nevertheless, the genomic architecture of both serotypes is most similar to that of FMDV. Indeed, a comparison of the derived protein sequences of ERV-1 shows that their identity is greatest to FMDV. In contrast, most ERV-2 proteins are more related to encephalomyocarditis virus (EMCV) proteins than they are to FMDV or ERV-1. These results place ERV-1 alongside FMDV in the aphthovirus genus of the picornavirus family and indicate that this virus may serve as a model system for examining the biology of FMDV.

Amino Acid Sequence↗
PubMed1997

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theiler's murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Amino Acid Sequence↗