Competition-colonization dynamics in an RNA virus

Abstract

During replication, RNA viruses rapidly generate diverse mutant progeny which differ in their ability to kill host cells. We report that the progeny of a single RNA viral genome diversified during hundreds of passages in cell culture and self-organized into two genetically distinct subpopulations that exhibited the competition-colonization dynamics previously recognized in many classical ecological systems. Viral colonizers alone were more efficient in killing cells than competitors in culture. In cells coinfected with both competitors and colonizers, viral interference resulted in reduced cell killing, and competitors replaced colonizers. Mathematical modeling of this coinfection dynamics predicted selection to be density dependent, which was confirmed experimentally. Thus, as is known for other ecological systems, biodiversity and even cell killing of virus populations can be shaped by a tradeoff between competition and colonization. Our results suggest a model for the evolution of virulence in viruses based on internal interactions within mutant spectra of viral quasispecies.

Fig. 1.

Genetic characterization of p200 and MARLS viruses. (A) Maximum likelihood reconstruction of consensus nucleotide sequences (nucleotides 1033 through 1154 and 1570 through 3853, see Materials and Methods) of populations before and after three sequential low-MOI infections (indicated by “p3d”). (B) Maximum likelihood reconstruction [nucleotides 1033 through 3853; residue numbering is as described in (10)] of biological clones. Clones are identified by passage number and clone number (e.g., 225c6). Consensus reference sequences at passages 0, 200 (p0, p200), and of MARLS virus are included. Confidence values higher than 80% are indicated.

Fig. 2.

Cell killing and cell killing-interference assays. (A and B) Cell killing capacity of viral populations and clones, measured as the time needed to kill 10⁴ BHK-21 cells as a function of the initial number of PFU. (A) MARLS clones: 240c2 (○) and 240c12 (Δ); p200 clones: 240c1 (▲) and 240c13 (×). (B) Population p200 (▪), population p200p5d, MARLS population (□). For each point, the average and standard deviation from three independent determinations are indicated. (C–E) Cell killing-interference assays. Number of infectious centers required by p200, MARLS, or the mixture of both viruses to kill 10⁴ BHK-21 cells in 9.5h; 2×, double dose of MARLS clone or population. Each bar represents the mean and standard deviation from triplicate assays. (C) p200 clone and MARLS clone correspond to 240c1 and 240c12, respectively; *The mean of the “Clone mix” is higher than the mean of the “MARLS clone” with marginal significance (P < 0.08, T-Student). (D) population mix indicates a mixture of MARLS population and p200 population. *The mean of the “population mix” is significantly higher (P < 0.05, T-Student) than the mean of the MARLS population. (E) C-S8p260p3d is the virus recovered by passage of C-S8c1 after 260 transfers in cell culture and 3 passages at low-MOI; this virus has a virulence similar to MARLS (10).

Fig. 3.

Model predictions and experimental results of virus competition experiments. The basic model of virus competition in cell culture was implemented and solved for the parameters listed in Table 1, to obtain relative fitness of MARLS compared to p200 which are plotted as a function of MOI (). Experimental fitness values were obtained from virus competition experiments. Two representative clones each of MARLS and p200 subpopulations were mixed at equal PFU, and challenged in competitions with variable initial MOI. Reference p200 and MARLS populations (p200p5d) were also mixed in direct competition at variable initial MOI. Relative fitness values obtained in each competition for both clones and populations are plotted against MOI (▲). Experimental measurements with standard errors (solid line) and model predictions (dashed line) were fitted by linear regression. Procedures for fitness value measurements and competition experiments are detailed in Materials and Methods and the SI Appendix.