Foot-and-mouth Disease Virus Populations Research Articles

Establishing an In Vitro System to Assess How Specific Antibodies Drive the Evolution of Foot-and-Mouth Disease Virus.

Viruses can evolve to respond to immune pressures conferred by specific antibodies generated after vaccination and/or infection. In this study, an in vitro system was developed to investigate the impact of serum-neutralising antibodies upon the evolution of a foot-and-mouth disease virus (FMDV) isolate. The presence of sub-neutralising dilutions of specific antisera delayed the onset of virus-induced cytopathic effect (CPE) by up to 44 h compared to the untreated control cultures. Continued virus passage with sub-neutralising dilutions of these sera resulted in a decrease in time to complete CPE, suggesting that FMDV in these cultures adapted to escape immune pressure. These phenotypic changes were associated with three separate consensus-level non-synonymous mutations that accrued in the viral RNA-encoding amino acids at positions VP266, VP280 and VP1155, corresponding to known epitope sites. High-throughput sequencing also identified further nucleotide substitutions within the regions encoding the leader (Lpro), VP4, VP2 and VP3 proteins. While association of the later mutations with the adaptation to immune pressure must be further verified, these results highlight the multiple routes by which FMDV populations can escape neutralising antibodies and support the application of a simple in vitro approach to assess the impact of the humoral immune system on the evolution of FMDV and potentially other viruses.

Into the Deep (Sequence) of the Foot-and-Mouth Disease Virus Gene Pool: Bottlenecks and Adaptation during Infection in Naïve and Vaccinated Cattle.

Foot-and-mouth disease virus (FMDV) infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail in natural host species. In this study, virus samples collected from vaccinated and non-vaccinated cattle up to 35 days post-experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle in substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection reflected the diversity inherited from the source virus (inoculum), whereas by 12 days post infection, dominant viruses were defined by newly acquired mutations. Mutations conferring recognized fitness gain occurred and were associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting distinct foci of infection within the nasopharyngeal mucosa. Subclinical infection in vaccinated cattle included very early bottlenecks associated with reduced diversity within virus populations. Viruses from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission is less likely to occur. This study improves upon previously published work by analyzing deep sequences of samples, allowing for detailed characterization of FMDV populations over time within multiple hosts.

Estimating viral bottleneck sizes for FMDV transmission within and between hosts and implications for the rate of viral evolution.

RNA viruses exist as populations of closely related genomes, characterized by a high diversity of low-frequency variants. As viral genomes from one population disperse to establish new sites of replication, the fate of these low-frequency variants depends to a large extent on the size of the founding population. Focusing on foot-and-mouth disease virus (FMDV) we conjecture that variants are more likely to be transmitted through wide bottlenecks, but more likely to approach fixation in new populations following narrow bottlenecks; therefore, the longer-term rate of accumulation of 'nearly neutral' variants at high frequencies is likely to be inversely related to the bottleneck size. We examine this conjecture in vivo by estimating bottleneck sizes relating 'parent' and 'daughter' populations observed at different scales ranging from within host to between host (within the same herd, and in different herds) using a previously established method. Within hosts, we find bottleneck sizes to range from 5 to 20 viral genomes between populations transmitted from the pharynx to the serum, and from 4 to 54 between serum and lesion populations. Between hosts, we find bottleneck sizes to range from 2 to 39, suggesting inter-host bottlenecks are of a similar size to intra-host bottlenecks. We establish a statistically significant negative relationship between the probability of genomic consensus level change and bottleneck size, and present a simple sampling model that captures this empirical relationship. We also present a novel in vitro experiment to investigate the impact of bottleneck size on the frequency of mutations within FMDV populations, demonstrate that variant frequency in a population increases more rapidly during small population passages, and provide evidence for positive selection during the passage of large populations.

Genetic stability of foot-and-mouth disease virus during long-term infections in natural hosts.

Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008–2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008–2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.

Lethal Mutagenesis of Foot-and-Mouth Disease Virus Involves Shifts in Sequence Space

Lethal mutagenesis or virus transition into error catastrophe is an antiviral strategy that aims at extinguishing a virus by increasing the viral mutation rates during replication. The molecular basis of lethal mutagenesis is largely unknown. Previous studies showed that a critical substitution in the foot-and-mouth disease virus (FMDV) polymerase was sufficient to allow the virus to escape extinction through modulation of the transition types induced by the purine nucleoside analogue ribavirin. This substitution was not detected in mutant spectra of FMDV populations that had not replicated in the presence of ribavirin, using standard molecular cloning and nucleotide sequencing. Here we selectively amplify and analyze low-melting-temperature cDNA duplexes copied from FMDV genome populations passaged in the absence or presence of ribovirin Hypermutated genomes with high frequencies of A and U were present in both ribavirin -treated and untreated populations, but the major effect of ribavirin mutagenesis was to accelerate the occurrence of AU-rich mutant clouds during the early replication rounds of the virus. The standard FMDV quasispecies passaged in the absence of ribavirin included the salient transition-modulating, ribavirin resistance mutation, whose frequency increased in populations treated with ribavirin. Thus, even nonmutagenized FMDV quasispecies include a deep, mutationally biased portion of sequence space, in support of the view that the virus replicates close to the error threshold for maintenance of genetic information.

Molecular Characterization of a Dual Inhibitory and Mutagenic Activity of 5-Fluorouridine Triphosphate on Viral RNA Synthesis. Implications for Lethal Mutagenesis

The basis for a dual inhibitory and mutagenic activity of 5-fluorouracil (5-FU) on foot-and-mouth disease virus (FMDV) RNA replication has been investigated with purified viral RNA-dependent RNA polymerase (3D) in vitro. 5-Fluorouridine triphosphate acted as a potent competitive inhibitor of VPg uridylylation, the initial step of viral replication. Peptide analysis by mass spectrometry has identified a VPg fragment containing 5-fluorouridine monophosphate (FUMP) covalently attached to Tyr3, the amino acid target of the uridylylation reaction. During RNA elongation, FUMP was incorporated in the place of UMP or CMP by FMDV 3D, using homopolymeric and heteropolymeric templates. Incorporation of FUMP did not prevent chain elongation, and, in some sequence contexts, it favored misincorporations at downstream positions. When present in the template, FUMP directed the incorporation of AMP and GMP, with ATP being a more effective substrate than GTP. The misincorporation of GMP was 17-fold faster opposite FU than opposite U in the template. These results in vitro are consistent with the mutational bias observed in the mutant spectra of 5-FU-treated FMDV populations. The dual mutagenic and inhibitory activity of 5-fluorouridine triphosphate may contribute to the effective extinction of FMDV by 5-FU through virus entry into error catastrophe.

Topology of evolving, mutagenized viral populations: quasispecies expansion, compression, and operation of negative selection.

BackgroundThe molecular events and evolutionary forces underlying lethal mutagenesis of virus (or virus extinction through an excess of mutations) are not well understood. Here we apply for the first time phylogenetic methods and Partition Analysis of Quasispecies (PAQ) to monitor genetic distances and intra-population structures of mutant spectra of foot-and-mouth disease virus (FMDV) quasispecies subjected to mutagenesis by base and nucleoside analogues.ResultsPhylogenetic and PAQ analyses have revealed a highly dynamic variation of intrapopulation diversity of FMDV quasispecies. The population diversity first suffers striking expansions in the presence of mutagens and then compressions either when the presence of the mutagenic analogue was discontinued or when a mutation that decreased sensitivity to a mutagen was selected. The pattern of mutations found in the populations was in agreement with the behavior of the corresponding nucleotide analogues with FMDV in vitro. Mutations accumulated at preferred genomic sites, and dn/ds ratios indicate the operation of negative (or purifying) selection in populations subjected to mutagenesis. No evidence of unusually elevated genetic distances has been obtained for FMDV populations approaching extinction.ConclusionPhylogenetic and PAQ analysis provide adequate procedures to describe the evolution of viral sequences subjected to lethal mutagenesis. These methods define the changes of intra-population structure more precisely than mutation frequencies and Shannon entropies. PAQ is very sensitive to variations of intrapopulation genetic distances. Strong negative (or purifying) selection operates in FMDV populations subjected to enhanced mutagenesis. The quantifications provide evidence that extinction does not imply unusual increases of intrapopulation complexity, in support of the lethal defection model of virus extinction.

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.

Genetic Lesions Associated with Muller's Ratchet in an RNA Virus

The molecular basis of Muller's ratchet has been investigated using the important animal pathogen foot-and-mouth disease virus (FMDV). Clones from two FMDV populations were subjected to serial plaque transfers (repeated bottleneck events) on host BHK-21 cells. Relative fitness losses were documented in 11 out of 19 clones tested. Small fitness gains were observed in three clones. One viral clone attained an extremely low plating efficiency, suggesting that accumulation of deleterious mutations had driven the virus near extinction. Nucleotide sequence analysis revealed unique genetic lesions in multiply transferred clones that had never been seen in FMDVs isolated in nature or subjected to massive infections in cell culture. In particular, a frequent internal polyadenylate extension has identified a mutational hot spot on the FMDV genome. Furthermore, amino acid residue substitutions in internal capsid sites which are severely restricted during FMDV evolution, amounted to half of capsid replace- ments in the transferred clones. In addition, a striking dominance of non-synonymous replacements fixed upon large population infections of FMDV was not observed upon serial plaque transfers. The nucleotide sequence of the entire genome of a severely debilitated clone suggests that very few mutations may be sufficient to drive FMDV near extinction. The results provide an account of the molecular basis of Muller's ratchet for an RNA virus, and insight into the types of genetic variants which populate the mutant spectra of FMDV quasispecies.

Modification of foot-and-mouth disease virus after serial passages in the presence of antiviral polyclonal sera

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, this virus has a high rate of mutation. It has been proposed that selection exerted by the host's antibodies could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant populations (Nr), after serial passages of cloned FMDV A 24 Cruzeiro strain on secondary monolayers of bovine fetal kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages under selective pressure, the virus population showed the following characteristics: (1) increased resistance to neutralization by APS; (2) altered electrophoretic mobility of structural viral proteins (VP1); (3) remarkable plaque size reduction, (4) a pronounced thermosensitivity (ts); and (5) decreased pathogenicity for mice, in both uncloned and cloned small plaque size populations. This indicates that FMDV populations under antibody pressure in vitro, have acquired, in addition to expected characteristics of natural FMDV variants (resistance to neutralization and altered viral structural proteins), phenotypic markers which correspond to attenuated, less virulent variants.

Multiple genetic variants arise in the course of replication of foot-and-mouth disease virus in cell culture

The genetic heterogeneity generated upon passage of foot-and-mouth disease virus (FMDV) in cell culture has been evaluated by T1-oligonucleotide fingerprinting of genomic RNA. Plaque-purified FMDV O-S7 and C-S8 were propagated by serial low multiplicity infections of BHK-21 (c-13) or IBRS-2 (c-26) cells. In independent parallel passage of the same virus, different oligonucleotide variations were fixed in the RNAS. T1-oligonucleotide fingerprinting of RNA from 34 individual viral clones derived from two passaged populations shows an extensive heterogeneity, with some mutations present in only one of the cloned genomes analyzed. Some FMDV variants are phenotypically distinct in that they yield increased progeny in infections of cell monolayers. From the number of variant sequences it can be estimated that each infectious RNA in the population differs in two to eight mutations from the average parental sequence. Thus, passaged FMDV populations consist of a pool of variants, an observation previously made with phage Qβ ( E. Domingo, D. Sabo, T. Taniguchi, and C. Weissmann, Cell 13, 735–744, 1978 ). The FMDV genome must be described. as a fluctuating distribution of sequences due to its high mutability. This may be the basis of the extensive genetic and antigenic diversity of this virus in nature.