Abstract
The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.
Highlights
Three quarters of the recently discovered human pathogens are viral, and most of those are RNA viruses [1]
Recombination can lead to an interspecies transmission event, as was believed to be the case with SARS-CoV, accumulation of point mutations may enable the coronaviruses to adapt to new host species [5,6,7]
Naturally-infected Bovine coronavirus (BCoV) samples are often difficult to adapt to growth in cell culture [20], it is essential to use naturally-infected samples rather than laboratory adapted virus strains because isolation in cell culture is likely to result in adaptive mutations not present in the naturally circulating virus
Summary
Three quarters of the recently discovered human pathogens are viral, and most of those are RNA viruses [1] Some of these emergent viruses, such as HIV and SARS coronavirus (SARSCoV), are capable of causing epidemics of human disease. RNA virus populations sustain high genetic diversity due to the low fidelity of their polymerase, short genome, high replication rates and large population size [2]. For this reason a single RNA virus population can consist of a multiplicity of slightly different genomes, sometimes referred to as a mutant spectra [3]. Recombination can lead to an interspecies transmission event, as was believed to be the case with SARS-CoV, accumulation of point mutations may enable the coronaviruses to adapt to new host species [5,6,7]
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