Abstract
Virus production still is a challenging issue in antigen manufacture, particularly with slow-growing viruses. Deep-sequencing of genomic regions indicative of efficient replication may be used to identify high-fitness minority individuals suppressed by the ensemble of mutants in a virus quasispecies. Molecular breeding of quasispecies containing colonizer individuals, under regimes allowing more than one replicative cycle, is a strategy to select the fittest competitors among the colonizers. A slow-growing cell culture-adapted hepatitis A virus strain was employed as a model for this strategy. Using genomic selection in two regions predictive of efficient translation, the internal ribosome entry site and the VP1-coding region, high-fitness minority colonizer individuals were identified in a population adapted to conditions of artificially-induced cellular transcription shut-off. Molecular breeding of this population with a second one, also adapted to transcription shut-off and showing an overall colonizer phenotype, allowed the selection of a fast-growing population of great biotechnological potential.
Highlights
IntroductionPopulation F0.05LA was predicted to contain very low-frequency high-fitness individuals suppressed by the quasispecies, and whose isolation by playing with multiplicity of infection (MOI) or population size was anticipated to be difficult[15,17]
Two genomic regions were selected as markers of the breeding values in terms of virus productivity of the different individuals of the hepatitis A virus (HAV) quasispecies
Recombinant strategies to produce high capsid yields have systematically failed[23,24,25,26], which may be related to a low level of expression due to its highly deoptimized codon usage[6,13]
Summary
Population F0.05LA was predicted to contain very low-frequency high-fitness individuals suppressed by the quasispecies, and whose isolation by playing with MOI or population size was anticipated to be difficult[15,17]. These high-fitness individuals were predicted to be good colonizers (faster than the suppressors due to an efficient IRES) and quite good competitors (more optimized codon usage than many of the suppressors). We took advantage of population F0.2LA which has a colonizer behavior in competition experiments with population F0.05LA under conditions of low cellular shut-off and low MOI6 This behavior is mostly related to its very efficient uncoating process[13], which ensures a much faster cellular cycle. A fast-growing population producing larger plaques and higher virus titers was selected, with a remarkable biotechnological added value
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