MH, NP, CE, and ED conceived and designed the experiments. MH performed the experiments. MH, JGA, NP, CE, and ED analyzed the data. MH, JGA, NP, and CE contributed viruses, plasmids, materials, and information. MH and ED wrote the paper.
¤a Current address: Centro Nacional de Biotecnología (CSIC), Cantoblanco, Madrid, Spain
The authors have declared that no competing interests exist.
The relationship between parasite fitness and virulence has been the object of experimental and theoretical studies often with conflicting conclusions. Here, we provide direct experimental evidence that viral fitness and virulence, both measured in the same biological environment provided by host cells in culture, can be two unrelated traits. A biological clone of foot-and-mouth disease virus acquired high fitness and virulence (cell killing capacity) upon large population passages in cell culture. However, subsequent plaque-to-plaque transfers resulted in profound fitness loss, but only a minimal decrease of virulence. While fitness-decreasing mutations have been mapped throughout the genome, virulence determinants—studied here with mutant and chimeric viruses—were multigenic, but concentrated on some genomic regions. Therefore, we propose a model in which viral virulence is more robust to mutation than viral fitness. As a consequence, depending on the passage regime, viral fitness and virulence can follow different evolutionary trajectories. This lack of correlation is relevant to current models of attenuation and virulence in that virus de-adaptation need not entail a decrease of virulence.
Virulence expresses the harm that parasites inflict upon their hosts. Many studies have addressed the basis of virulence and its effect on host and parasite survival. It has generally been accepted that one of the components of parasite virulence is fitness, or the capacity of the parasite to multiply in its host. Some models have equated virulence with fitness. In the present study, we use foot-and-mouth disease virus (FMDV) to document that virulence and fitness—measured in the same biological environment provided by cells in culture—can be unrelated traits. This has been achieved by multiplying the virus in a manner that mutations accumulated in its genome. Mutations decreased fitness dramatically, but not virulence. Chimeric and mutant viruses were constructed to show that virulence is influenced by only some of the FMDV genes, while fitness is influenced by the entire genome. For this reason, virulence is more robust (“resistant”) than fitness to the effects of deleterious mutations. The fact that virulence can be unrelated to fitness has implications for the design of anti-viral vaccines because it suggests that it may be possible to design high fitness, low virulence strains to stimulate the host immune response. Furthermore, in modelling studies it cannot be assumed that virulence is equal to fitness.
The relationship between fitness and virulence is an unsettled question, and sometimes fitness is considered a component of the virulence phenotype of parasites. RNA viruses are ideal systems to address this important question because of their high mutability and fecundity, which result in a potential for rapid evolution, and also because of the availability of quantitative fitness and virulence assays.
RNA viruses replicate as complex and dynamic mutant spectra, termed viral quasispecies. Key to quasispecies dynamics are mutation rates in the range of 10−3 to 10−5 substitutions per nucleotide copied, and competition among continuously arising variant genomes [
An understanding of the consequences of fitness variation for viral virulence is a key question for viral pathogenesis and evolution. Here, we approach this issue with FMDV, an important viral pathogen in veterinary medicine [
In the present study, we define
The results suggest that fitness is very vulnerable to mutation in any genomic region. In contrast, because of the involvement of several (but not all) viral genes in virulence, and the redundant effect of three 2C substitutions, virulence is a more robust phenotypic trait than fitness, and less vulnerable to accumulation of mutations. Therefore, we provide direct evidence that viral fitness and capacity to kill cells can (in some cases) be unrelated traits. Furthermore, the relationship between fitness and virulence, of being either linked or unrelated traits, depends on the evolutionary history of the virus. This observation has implications for viral pathogenesis, and sheds light on models of virulence proposed on the basis of theoretical and experimental studies with cellular organisms.
Several biological clones and populations were obtained by passaging FMDV biological clone C-S8c1 [
C-S8c1 is the parental, reference biological clone of FMDV [
Values of Fitness and Virulence of FMDV Clones and Populations Analyzed in the Present Study
The capacity of
(A) Time needed by C-S8c1, REDpt60,
The regression line including
(B) Time needed by C-S8c1, C-S8c1p113,
Virulence and Progeny Production by Chimeric C-S8c1 (pMT28)/
The comparison of the consensus nucleotide sequence of the
The FMDV C-S8c1 genome (8,115 residues excluding the internal poly(C) and the 3′ poly(A)) composed of the 5′ and 3′ UTRs (lines) and coding regions (boxes), which include protease L, structural proteins (VP4, VP2, VP3, and VP1), and non-structural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D). Genomic regions are based in [
The C-S8c1 and
Time needed to kill 104 BHK-21 cells as a function of the initial PFU of the following viruses:
(A) C-S8c1, C-S8c1(pMT28),
(B) C-S8c1(pMT28),
(C) C-S8c1(pMT28),
(D) C-S8c1(pMT28),
Each value represents the mean and standard deviation from triplicate assays. The viruses analyzed are described in
Amino acid substitutions in human rhinovirus protein 2C promoted cytopathology for mouse L cells [
Time needed to kill 104 BHK-21 cells as a function of the initial number of PFU of the following viruses:
(A) C-S8c1(pMT28),
(B) C-S8c1(pMT28),
Each value represents the mean and standard deviation from triplicate assays. The viruses analyzed are described in
Thus, a virus that evolves towards low fitness levels due to the operation of Muller's ratchet may nevertheless maintain its capacity to kill the same cells in which it displays low fitness. In FMDV, the enhanced capacity to kill BHK-21 cells was multigenic, including participation of non-structural protein 2C with three amino acid substitutions acting in a redundant fashion. In conclusion, the results provide a molecular interpretation of why fitness and virulence of an animal virus can follow disparate evolutionary trajectories, culminating in two unrelated traits.
The capacity of a virus to kill cells is probably influenced by several steps in the virus life cycle, including receptor affinity (which may trigger signalling pathways and alter cell functions) and intracellular viral replication that may lead to metabolic alterations such as transcriptional or translational shut-off [
Replicative fitness is, however, but one of several factors which influence the progression of a viral infection in vivo. In a comparative analysis, R5-tropic and X4-tropic clones of HIV-1 showed similar replication capacity in mitogen-activated T cells. However, X4 clones were transferred more efficiently than R5 clones from dendritic cells to CD4(+) T cells, a fact that can contribute to the competitive advantage of X4 viruses in AIDS patients [
The results with FMDV clones H5 have documented that both fitness-enhancing and virulence-enhancing mutations can be incorporated in the viral genome in such a fashion that subsequent fitness-decreasing mutations associated with bottleneck (plaque-to-plaque) transfers produce only minimal effects on virulence (
The comparative analysis of FMDV clones and populations shows that shifts in virulence can occur even through the evolution of a single viral clone (C-S8c1), with its restricted genetic diversity prompted by different replication regimes in the same host cells, which also have a clonal origin (see
It must be emphasized that fitness and virulence are relative values that pertain to a defined physical and biological environment. Virulence determinants of FMDV, identified here for BHK-21 cells, need not apply to virulence for the natural animal hosts of FMDV [
Most current definitions of virulence include both the ability of the pathogen to multiply and to cause harm to its host; some authors, however, assume a direct relationship between fitness and capacity to produce disease [
The invariance of BHK-21 cells in the course of serial cytolytic passages of FMDV is in contrast with the parallel system consisting of BHK-21 cells persistently infected with FMDV C-S8c1 [
Our comparison of FMDV clones did not provide evidence of clones with high fitness and low virulence, which, with regard to natural hosts, is an aim of biomedicine to obtain vaccine strains. Yet, the existence of specific mutations that differentially affect fitness and virulence opens the way to engineer candidate vaccine strains unable to kill the host, while maintaining replicative competence. Virulence is, however, a feature of the host–parasite relationship [
The BHK-21 cells used in the present study were cloned by end-point dilution, followed by preparation of a cell stock from a single cell; they were passaged a maximum of 30 times before being used for FMDV infection [
The capacity of FMDV to kill BHK-21 cells was measured as previously described [
The relative fitness of FMDV
Viral RNA was extracted by treatment with Trizol as previously described [
Chimeric viruses containing selected regions of
To obtain FMDV C-S8c1 containing the mutations found in gene 2C of
DNA from pMT28 or its recombinant and mutant derivatives was linearized with Nde I and transcribed with SP6 RNA polymerase as previously described [
Consensus genomic nucleotide sequences of FMDV clones were obtained by RT-PCR amplification of virion RNA using specific primers [
aThe origin of the FMDVs is described in
bEstimated relative virulence as the value extrapolated from the regression at 0 h postinfection.
cRegression lines with the data of 12 h to 48 h postinfection were obtained using the program Excel from the Microsoft Office package.
(46 KB DOC)
aThe first letter corresponds to the nucleotide found in the parental FMDV C-S8c1, and the number gives the nucleotide position in the C-S8c1 genome [
bThe first amino acid is the one found in C-S8c1; amino acid residues are numbered independently for each protein. -, synonymous mutation; *, amino acid change in invariant positions of the FMDV genome according to [
cH clone in which the mutation appears for the first time [
(88 KB DOC)
aThe residue numbering of the FMDV genome is as in [
bNucleotide replacements required for mutagenesis are underlined.
(44 KB DOC)
The GenBank accession numbers for the C-S8c1,
We thank M. Dávila for expert technical assistance and A. Arias for the critical reading of the manuscript.
¤b Current address: Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles AIDS Institute, Los Angeles, California, United States of America
baby hamster kidney 21
foot-and-mouth disease virus
human immunodeficiency virus type 1
multiplicity of infection
plaque-forming units
reverse transcription PCR