Molecular basis for a lack of correlation between viral fitness and cell killing capacity

Abstract

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.

Figure 1. Schematic Representation of the Origin of the FMDVs Used in the Present Study

C-S8c1 is the parental, reference biological clone of FMDV [19]. Biological clones are depicted as filled squares and uncloned populations as empty circles; p followed by a number indicates passage number. Large grey arrows indicate high MOI passages (1 to 5 PFU/cell); thin arrows describe the isolation of biological clones (virus from individual plaques) after dilution of virus and plating on BHK-21 cell monolayers; thick black arrows indicate the selection of mutant RED [20] and MARLS [21], which are resistant to neutralization by monoclonal antibody SD6; large empty arrows indicate low MOI passages carried out to derive the standard C-S8p260p3d genome from a bipartite, segmented form of the FMDV genome termed C-S8p260 [22]. The origin of viruses used in the present study is detailed in Materials and Methods.

Figure 2. Killing of BHK-21 Cells by FMDVs

(A) Time needed by C-S8c1, REDpt60, , C-S8p260p3d, and MARLS to kill 10⁴ BHK-21 cells as a function of the initial number of infectious units (PFU) added. The virulence assay is described in Materials and Methods. Each value represents the mean and standard deviation from triplicate assays. Inset: Relative fitness as a function of relative virulence values of FMDVs. The regression (discontinuous) line defined by C-S8c1, REDpt60, C-S8p260p3d, and MARLS is y = 3.026Ln(x) – 1.1028; R ² = 0.9721. The regression line including is y = 3.1458Ln(x) – 3.5111; R ² = 0.8507 (not drawn). (B) Time needed by C-S8c1, C-S8c1p113, , and to kill 10⁴ BHK-21 cells as a function of the initial number of PFU. Each value represents the mean and standard deviation from triplicate assays. The viruses analyzed are described in Materials and Methods, and their evolutionary relationship is depicted in Figure 1. Virulence values are given in Tables 1, 2, and S1.

Figure 3. Amino Acid Substitutions Found in the FMDV Genome as Compared to FMDV C-S8c1

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 [14] and references therein; VPg is the protein (3B) covalently linked to the 5′ end of the RNA, poly(C) is the internal polyribocytidylate tract, and poly(A) is the 3′ terminal polyadenylate tract. The FMDV C-S8c1- and -coding regions are represented in white and blue, respectively. Amino acids in that differ from the corresponding ones in C-S8c1 are indicated in red. Replacements found also in the genome are encircled. Numbering of amino acids for each individual protein is as in [11].

Figure 4. Scheme of the FMDV Genome, Chimeric Viruses, and 2C Mutants

The C-S8c1 and regulatory regions are represented as black and blue lines, respectively; the corresponding coding regions are represented as white and blue boxes, respectively. pMT28 refers to the plasmid that encodes infectious RNA transcripts with the C-S8c1 nucleotide sequence [22]. Chimeric viruses are colored blue and named with the first and the last nucleotide that correspond to the genomic region. Chimera /2C-3A(pMT28) includes 2C, 3A, and parts of 2B and 3B of pMT28 in the background of residues 436 to 7427 of . Amino acid substitutions in protein 2C are indicated in red. The residue numbering of the FMDV genome is as in [11]. Procedures for the construction of chimeric viruses and 2C mutants are described in Materials and Methods.

Figure 5. Killing of BHK-21 Cells by Recombinant FMDV Chimeras

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), , pMT28/ (436-2046), pMT28/ (2046–3760), and pMT28/ (436-3760). (B) C-S8c1(pMT28), , pMT28/ (3760–5839), pMT28/ (5839–7427), and pMT28/ (3760–7427). (C) C-S8c1(pMT28), , pMT28/ (2046–7427), and pMT28/ (436-7427). (D) C-S8c1(pMT28), , pMT28/ (436-7427), and /2C-3A(pMT28). Each value represents the mean and standard deviation from triplicate assays. The viruses analyzed are described in Materials and Methods; C-S8c1(pMT28) is C-S8c1 expressed as an infectious transcript and rescued by cell transfection. Virulence values are given in Tables 2 and S1.

Figure 6. Killing of BHK-21 Cells by FMDV Mutants

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), , pMT28 (SN), pMT28 (TA), and pMT28 (QH). (B) C-S8c1(pMT28), _, and pMT28 (SN, TA, QH). Each value represents the mean and standard deviation from triplicate assays. The viruses analyzed are described in Materials and Methods. Virulence values are given in Tables 2 and S1.