Foot-and-mouth Disease Virus Clones Research Articles

Selection and characterization of an acid-resistant mutant of serotype O foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.

Biological Effect of Muller's Ratchet: Distant Capsid Site Can Affect Picornavirus Protein Processing

Repeated bottleneck passages of RNA viruses result in accumulation of mutations and fitness decrease. Here, we show that clones of foot-and-mouth disease virus (FMDV) subjected to bottleneck passages, in the form of plaque-to-plaque transfers in BHK-21 cells, increased the thermosensitivity of the viral clones. By constructing infectious FMDV clones, we have identified the amino acid substitution M54I in capsid protein VP1 as one of the lesions associated with thermosensitivity. M54I affects processing of precursor P1, as evidenced by decreased production of VP1 and accumulation of VP1 precursor proteins. The defect is enhanced at high temperatures. Residue M54 of VP1 is exposed on the virion surface, and it is close to the B-C loop where an antigenic site of FMDV is located. M54 is not in direct contact with the VP1-VP3 cleavage site, according to the three-dimensional structure of FMDV particles. Models to account for the effect of M54 in processing of the FMDV polyprotein are proposed. In addition to revealing a distance effect in polyprotein processing, these results underline the importance of pursuing at the biochemical level the biological defects that arise when viruses are subjected to multiple bottleneck events.

The 30th anniversary of quasispecies

The meeting on ‘Quasispecies: past, present and future’ took place between 17 and 18 November 2008, in Barcelona, Spain, and was organized by J. Gomez, C. Lopez‐Galindez, M.A. Martinez & A. Mas. ![][1] A meeting was held in Barcelona, Spain, in November 2008 to celebrate the 30th anniversary of the publication of the article that described the extensive genetic heterogeneity of bacteriophage Qβ (Domingo et al , 1978), which is considered to mark the beginning of experimental studies on viral quasispecies. This meeting was held at the impressive fifteenth century building of the ancient Hospital de la Santa Creu in the old town of Barcelona, which is now the headquarters of the Institut d'Estudis Catalans, and was attended by C. Weissmann (Jupiter, FL, USA), M. Billeter (Zurich, Switzerland) and E. Domingo (Madrid, Spain), who were three early protagonists of the phage Qβ work at the University of Zurich in the 1970s. Several speakers presented their results on the theoretical aspects of the population dynamics of cells and viruses, the clinical implications of quasispecies, and extensions of the quasispecies concept to cellular genes and prions. The meeting was introduced by A. Mas (Albacete, Spain), who reflected on the increasing impact that quasispecies have had in the scientific literature over the past three decades, and quoted some of the key references on viral quasispecies (Martell et al , 1992; Meyerhans et al , 1989; Najera et al , 1995; Vignuzzi et al , 2006; for a historical review of the impact of quasispecies in virology, see Holland, 2006). The scientific presentations were opened by Weissmann and Domingo, who were the last and first authors of the 1978 paper, respectively. Their talks conveyed the scientific atmosphere of the 1970s—when molecular biology was carried out with few recombinant‐DNA techniques—to a young audience. Nucleic‐acid sequencing was in … [1]: /embed/graphic-1.gif

Repeated Bottleneck Transfers Can Lead to Non-cytocidal Forms of a Cytopathic Virus: Implications for Viral Extinction

Several biological subclones of a biological clone of foot-and-mouth disease virus (FMDV) have been subjected to many plaque-to-plaque (serial bottleneck) transfers in cell culture. At transfer 190 to 409, clones underwent a transition towards a non-cytolytic (NC) phenotype in which the virus was unable to produce plaques, representing at least a 140-fold reduction in specific infectivity relative to the parental biological clone. NC clones, however, were competent in RNA replication and established a persistent infection in cell culture without an intervening cytolytic phase. In one clone, the transition to the NC phenotype was associated with the elongation of an internal oligodenylate tract that precedes the second functional AUG translation initiation codon. The pattern of mutations and their distribution along the FMDV genome of the clones subjected to serial bottleneck transfers were compared with the pattern of mutations in FMDV clones subjected to large population passages. Both the corrected ratios of non-synonymous to synonymous mutations and some specific mutations in coding and non-coding regions suggest participation of positive selection during large population passages and not during bottleneck transfers. Some mutations in the clones that attained the NC phenotype were located in genomic regions affecting the capacity of FMDV to kill BHK-21 cells. The resistance to extinction of clones subjected to plaque-to-plaque transfers marks a striking contrast with regard to the ease of extinction mediated by lethal mutagenesis. The results document a major phenotypic transition of a virus as a result of serial bottleneck events.

Quasispecies dynamics and RNA virus extinction

The extinction of foot-and-mouth disease virus (FMDV) is strongly influenced by mutation rates, types of mutations, relative viral fitness and virus population regimens during infection. Here we review experimental results and theoretical models that describe a contrast between the effective extinction of FMDV subjected to increased mutagenesis, and the remarkable resistance to extinction of the same and related FMDV clones subjected to serial bottleneck events. The results suggest procedures to master key parameters to develop effective antiviral strategies based on virus entry into error catastrophe.

Monitoring Sequence Space as a Test for the Target of Selection in Viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.

Fitness Increase of Memory Genomes in a Viral Quasispecies

Viral quasispecies may contain a subset of minority genomes that reflect those genomic sequences that were dominant at an early phase of quasispecies evolution. Such minority genomes are referred to as memory in viral quasispecies. A memory marker previously characterized in foot-and-mouth disease virus (FMDV) is an internal oligoadenylate tract of variable length that became dominant upon serial plaque-to-plaque transfers of FMDV clones. During large population passages, genomes with internal oligoadenylate were outcompeted by wild-type revertants but remained in the mutant spectra as memory genomes. Here, we report a quantification of relative fitness of several FMDV clones, harboring internal oligoadenylate tracts of different length, and that were retrieved at early or late times (passage number) after implementation of memory. The results show that for any given length range of the oligoadenylate, maintenance in memory resulted in an increase in relative fitness, comparable to the increase undergone by the entire population. The fitness increase is in agreement with the Red Queen hypothesis, and implies a replicative memory mechanism. Thus, permanence of memory genomes may be a source of high fitness variants despite their initial low fitness, and despite having remained hidden in mutant spectra. This reinforces the interest of diagnosing minority genomes during chronic human and animal viral infections.

Fitness Increase of Memory Genomes in a Viral Quasispecies

Viral quasispecies may contain a subset of minority genomes that reflect those genomic sequences that were dominant at an early phase of quasispecies evolution. Such minority genomes are referred to as memory in viral quasispecies. A memory marker previously characterized in foot-and-mouth disease virus (FMDV) is an internal oligoadenylate tract of variable length that became dominant upon serial plaque-to-plaque transfers of FMDV clones. During large population passages, genomes with internal oligoadenylate were outcompeted by wild-type revertants but remained in the mutant spectra as memory genomes. Here, we report a quantification of relative fitness of several FMDV clones, harboring internal oligoadenylate tracts of different length, and that were retrieved at early or late times (passage number) after implementation of memory. The results show that for any given length range of the oligoadenylate, maintenance in memory resulted in an increase in relative fitness, comparable to the increase undergone by the entire population. The fitness increase is in agreement with the Red Queen hypothesis, and implies a replicative memory mechanism. Thus, permanence of memory genomes may be a source of high fitness variants despite their initial low fitness, and despite having remained hidden in mutant spectra. This reinforces the interest of diagnosing minority genomes during chronic human and animal viral infections.

Modeling viral genome fitness evolution associated with serial bottleneck events: evidence of stationary states of fitness.

Evolution of fitness values upon replication of viral populations is strongly influenced by the size of the virus population that participates in the infections. While large population passages often result in fitness gains, repeated plaque-to-plaque transfers result in average fitness losses. Here we develop a numerical model that describes fitness evolution of viral clones subjected to serial bottleneck events. The model predicts a biphasic evolution of fitness values in that a period of exponential decrease is followed by a stationary state in which fitness values display large fluctuations around an average constant value. This biphasic evolution is in agreement with experimental results of serial plaque-to-plaque transfers carried out with foot-and-mouth disease virus (FMDV) in cell culture. The existence of a stationary phase of fitness values has been further documented by serial plaque-to-plaque transfers of FMDV clones that had reached very low relative fitness values. The statistical properties of the stationary state depend on several parameters of the model, such as the probability of advantageous versus deleterious mutations, initial fitness, and the number of replication rounds. In particular, the size of the bottleneck is critical for determining the trend of fitness evolution.

A single amino acid substitution in nonstructural protein 3A can mediate adaptation of foot-and-mouth disease virus to the guinea pig.

The genetic changes selected during the adaptation of a clonal population of foot-and-mouth disease virus (FMDV) to the guinea pig have been analyzed. FMDV clone C-S8c1 was adapted to the guinea pig by serial passage in the animals until secondary lesions were observed. Analysis of the virus directly recovered from the lesions developed by the animals revealed the selection of variants with two amino acid substitutions in nonstructural proteins, I(248)-->T in 2C and Q(44)-->R in 3A. On further passages, an additional mutation, L(147)-->P, was selected in an important antigenic site located in the G-H loop of capsid protein VP1. The amino acid substitution Q(44)-->R in 3A, either alone or in combination with the replacement I(248)-->T in 2C, was sufficient to give FMDV the ability to produce lesions. This was shown by using infectious transcripts which generated chimeric viruses with the relevant amino acid substitutions. Clinical symptoms produced by the artificial chimeras were similar to those produced by the naturally adapted virus. These results obtained with FMDV imply that one or very few replacements in nonstructural viral proteins, which should be within reach of the mutant spectra of replicating viral quasispecies, may result in adaptation of a virus to a new animal host.

Response of foot-and-mouth disease virus to increased mutagenesis: influence of viral load and fitness in loss of infectivity.

Passage of foot-and-mouth disease virus (FMDV) in cell culture in the presence of the mutagenic base analog 5-fluorouracil or 5-azacytidine resulted in decreases of infectivity and occasional extinction of the virus. Low viral loads and low viral fitness enhanced the frequency of extinction events; this finding was shown with a number of closely related FMDV clones and populations differing by up to 10(6)-fold in relative fitness in infections involving either single or multiple passages in the absence or presence of the chemical mutagens. The mutagenic treatments resulted in increases of 2- to 6.4-fold in mutation frequency and up to 3-fold in mutant spectrum complexity. The largest increase observed corresponded to the 3D (polymerase)-coding region, which is highly conserved in nonmutagenized FMDV populations. As a result, nucleotide sequence heterogeneity for the 3D-coding region became very similar to that for the variable VP1-coding region in FMDVs multiply passaged in the presence of chemical mutagens. The results suggest that strategies to combine reductions of viral load and viral fitness could be effectively associated with extinction mutagenesis as a potential new antiviral strategy.

Multiple molecular pathways for fitness recovery of an RNA virus debilitated by operation of Muller’s ratchet

Repeated bottleneck passages of RNA viruses result in fitness losses due to accumulation of deleterious mutations. We have analysed the molecular events underlying fitness recovery of a highly debilitated foot- and-mouth disease virus (FMDV) clone, upon serial passage in BHK-21 cells. The debilitated clone included an unusual, internal polyadenylate extension preceding the second functional AUG initiation codon, and a number of additional mutations scattered throughout the genome. Comparison of entire genomic nucleotide sequences in the course of passaging documented that loss of the internal polyadenylate was the first event in the process of fitness recovery. Further increases of fitness were associated with very few true reversions and with the accumulation of additional mutations affecting non-coding and coding regions. Remarkably, four biological subclones of the same debilitated FMDV clone gained fitness through three separate molecular pathways regarding correction of the internal polyadenylate: (i) a true reversion to yield the wild-type sequence at the second functional AUG; (ii) a shortening of the internal polyadenylate tract; or (iii) a deletion of 69 residues spanning the site of the polyadenylate extension. The results document that an RNA virus can find multiple pathways to reach alternative high fitness peaks on the fitness landscape.

Evolution subverting essentiality: dispensability of the cell attachment Arg-Gly-Asp motif in multiply passaged foot-and-mouth disease virus.

Aphthoviruses use a conserved Arg-Gly-Asp triplet for attachment to host cells and this motif is believed to be essential for virus viability. Here we report that this triplet-which is also a widespread motif involved in cell-to-cell adhesion-can become dispensable upon short-term evolution of the virus harboring it. Foot-and-mouth disease virus (FMDV), which was multiply passaged in cell culture, showed an altered repertoire of antigenic variants resistant to a neutralizing monoclonal antibody. The altered repertoire includes variants with substitutions at the Arg-Gly-Asp motif. Mutants lacking this sequence replicated normally in cell culture and were indistinguishable from the parental virus. Studies with individual FMDV clones indicate that amino acid replacements on the capsid surface located around the loop harboring the Arg-Gly-Asp triplet may mediate in the dispensability of this motif. The results show that FMDV quasispecies evolving in a constant biological environment have the capability of rendering totally dispensable a receptor recognition motif previously invariant, and to ensure an alternative pathway for normal viral replication. Thus, variability of highly conserved motifs, even those that viruses have adapted from functional cellular motifs, can contribute to phenotypic flexibility of RNA viruses in nature.

Evolution of a persistent aphthovirus in cytolytic infections: partial reversion of phenotypic traits accompanied by genetic diversification.

Foot-and-mouth disease virus (FMDV) shows a dual potential to be cytolytic or to establish persistent infections in cell culture. FMDV R100, a virus rescued after 100 passages of carrier BHK-21 cells persistently infected with FMDV clone C-S8c1, showed multiple genetic and phenotypic alterations relative to the parental clone C-S8c1. Several FMDV R100 populations have been subjected to 100 serial cytolytic infections in BHK-21 cells, and the reversion of phenotypic and genetic alterations has been analyzed. An extreme temperature sensitivity of R100 reverted totally or partially in some passage series but not in others. The small-plaque morphology reverted to normal size in all cases. The hypervirulence for BHK-21 cells did not revert, and even showed an increase, upon cytolytic passage. Most of the mutations that had been fixed in the R100 genome during persistence did not revert in the course of cytolytic passages, but the extended polyribocytidylate tract of R100 (about 460 residues, versus 290 in C-S8c1) decreased dramatically in length, to the range of 220 to 260 residues in all passage series examined. In passages involving very large viral populations, a variant with two amino acid substitutions (L-144-->V and A-145-->P) next to the highly conserved Arg-Gly-Asp (RGD motif; positions 141 to 143) within the G-H loop of capsid protein VP1 became dominant. A clonal analysis allowed isolation of a mutant with the single replacement A-145-->P. Viral production and growth competition experiments showed the two variants to have a fitness very close to that of the parental virus. The results provide evidence that the repertoire of variants that could potentially become dominant in viral quasispecies may be influenced by the population size of the evolving virus. The net results of a series of persistent-infection passages followed by a series of cytolytic passages was progressive genomic diversification despite reversion or stasis of phenotypic traits. Implications for the evolution of RNA viruses are discussed.

Distinct repertoire of antigenic variants of foot-and-mouth disease virus in the presence or absence of immune selection.

Antigenic variants of foot-and-mouth disease virus (FMDV) were generated and frequently became dominant in clonal populations of FMDV (clone C-S8c1) grown in the absence of anti-FMDV antibodies. We have now passaged eight samples of the same FMDV clone in the presence of a limited amount of neutralizing polyclonal antibodies directed to the major antigenic site A of capsid protein VP1. Complex populations of variants showing increased resistance to polyclonal sera and to site A-specific monoclonal antibodies were selected. Some populations exhibited marked decreases in viral fitness. Multiple amino acid replacements within site A--and also elsewhere in VP1--accumulated upon passage of the virus in either the absence or the presence of neutralizing antibodies. However, antigenically critical replacements at one position in site A occurred repeatedly in FMDV passaged under antibody selection, but they were never observed in many passages carried out either in the absence of antiviral antibodies or in the presence of an irrelevant antiviral serum. Thus, even though antigenic variation of FMDV can occur in the absence or presence of immune selection, critical replacements which lead to important changes in antigenic specificity were observed only as a result of selection by neutralizing antibodies.