Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population

Abstract

An RNA virus population does not consist of a single genotype; rather, it is an ensemble of related sequences, termed quasispecies. Quasispecies arise from rapid genomic evolution powered by the high mutation rate of RNA viral replication. Although a high mutation rate is dangerous for a virus because it results in nonviable individuals, it has been hypothesized that high mutation rates create a 'cloud' of potentially beneficial mutations at the population level, which afford the viral quasispecies a greater probability to evolve and adapt to new environments and challenges during infection. Mathematical models predict that viral quasispecies are not simply a collection of diverse mutants but a group of interactive variants, which together contribute to the characteristics of the population. According to this view, viral populations, rather than individual variants, are the target of evolutionary selection. Here we test this hypothesis by examining the consequences of limiting genomic diversity on viral populations. We find that poliovirus carrying a high-fidelity polymerase replicates at wild-type levels but generates less genomic diversity and is unable to adapt to adverse growth conditions. In infected animals, the reduced viral diversity leads to loss of neurotropism and an attenuated pathogenic phenotype. Notably, using chemical mutagenesis to expand quasispecies diversity of the high-fidelity virus before infection restores neurotropism and pathogenesis. Analysis of viruses isolated from brain provides direct evidence for complementation between members in the quasispecies, indicating that selection indeed occurs at the population level rather than on individual variants. Our study provides direct evidence for a fundamental prediction of the quasispecies theory and establishes a link between mutation rate, population dynamics and pathogenesis.

Figure 1

A restricted quasispecies of poliovirus is less neuropathogenic. a–b, Percentage of mice surviving intramuscular injection of different doses (10⁷, 10⁸, 10⁹ PFU) of the narrow G64S (a) or the expanded G64S^eQS populations (b) as compared to wildtype (WT, open symbols, only one dose, 10⁷ PFU is shown). The number of mice per group was 20 (n=20). The differences observed between WT (10⁷ PFU) and G64S (10⁷, 10⁸, 10⁹ PFU) or the expanded G64S^eQS (10⁷ PFU) are statistically significant (P<0.001). In contrast, no statistically significant difference was observed between WT (10⁷ PFU) and G64S^eQS (10⁸ PFU) (p>0.5). c, Calculation of LD₅₀ values for each viral stock using the Reed and Muench method.

Figure 2

Genomic diversity in a quasispecies is critical in viral tissue tropism and pathogenesis. a–b, Virus titers in PFU/gram from tissue of mice infected intravenously with the wildtype virus population (squares), the narrow G64S quasispecies (circles in a) or the artificially expanded G64S^eQS quasispecies (circles in b). Mean values ± s.d. of 3 experiments are shown.

Figure 2

Genomic diversity in a quasispecies is critical in viral tissue tropism and pathogenesis. a–b, Virus titers in PFU/gram from tissue of mice infected intravenously with the wildtype virus population (squares), the narrow G64S quasispecies (circles in a) or the artificially expanded G64S^eQS quasispecies (circles in b). Mean values ± s.d. of 3 experiments are shown.

Figure 3

Cooperative interactions among members of the virus population link quasispecies diversity with pathogenesis. a, Subpopulations of viruses isolated from brain of infected mice cannot re-establish CNS infection if the diversity of the quasispecies is restricted. Virus titers (PFU/g) from muscle, brain and spinal cord of mice infected intravenously for 4 days with 10⁷ PFU viruses isolated from brains of infected animals with wildtype^b, G64S^b, G64s^eQS-b. On top, schematic representation of the protocol of re-inoculation is shown. b, Neurotropic virus populations facilitate entry and replication of a non-neurotropic virus into the CNS. i) schematic representation of an in vivo complementation experiment. G64S^Sac is a narrow quasispecies carrying a higher fidelity polymerase (G64S allele) and a silent mutation that introduces a Sac I site at nucleotide 1906 within the capsid region. This neutral genetic marker can be used as “bar-code”. Mice were infected intravenously with either G64S^Sac alone (2x10⁸ PFU per animal) or co-injected with wildtype (WT) or G64S^eQS at 1:1 ratios (10⁸ PFU each virus per animal). Viruses were re-isolated from brain tissues, through infection of HeLa cells, and their RNA was analyzed by RT-PCR. ii) All PCR products were digested with Sac I prior agarose gel electrophoresis analysis. DNA obtained from wildtype or G64S^eQS viruses are not digested by Sac I (~3 kb fragment); whereas the PCR product from the G64S^Sac virus generated two smaller bands (~1450 and 1350 kb) when digested with Sac I. On top of lanes virus injected into each mouse are indicated. Each lane corresponds to one infected mouse. Arrows on the left indicate full-length RT-PCR products and Sac1 digested PCR fragments. DNA markers (kb) are shown (M).

Figure 3

Cooperative interactions among members of the virus population link quasispecies diversity with pathogenesis. a, Subpopulations of viruses isolated from brain of infected mice cannot re-establish CNS infection if the diversity of the quasispecies is restricted. Virus titers (PFU/g) from muscle, brain and spinal cord of mice infected intravenously for 4 days with 10⁷ PFU viruses isolated from brains of infected animals with wildtype^b, G64S^b, G64s^eQS-b. On top, schematic representation of the protocol of re-inoculation is shown. b, Neurotropic virus populations facilitate entry and replication of a non-neurotropic virus into the CNS. i) schematic representation of an in vivo complementation experiment. G64S^Sac is a narrow quasispecies carrying a higher fidelity polymerase (G64S allele) and a silent mutation that introduces a Sac I site at nucleotide 1906 within the capsid region. This neutral genetic marker can be used as “bar-code”. Mice were infected intravenously with either G64S^Sac alone (2x10⁸ PFU per animal) or co-injected with wildtype (WT) or G64S^eQS at 1:1 ratios (10⁸ PFU each virus per animal). Viruses were re-isolated from brain tissues, through infection of HeLa cells, and their RNA was analyzed by RT-PCR. ii) All PCR products were digested with Sac I prior agarose gel electrophoresis analysis. DNA obtained from wildtype or G64S^eQS viruses are not digested by Sac I (~3 kb fragment); whereas the PCR product from the G64S^Sac virus generated two smaller bands (~1450 and 1350 kb) when digested with Sac I. On top of lanes virus injected into each mouse are indicated. Each lane corresponds to one infected mouse. Arrows on the left indicate full-length RT-PCR products and Sac1 digested PCR fragments. DNA markers (kb) are shown (M).