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. 2016 Jul:41:233-239.
doi: 10.1016/j.meegid.2016.04.017. Epub 2016 Apr 17.

Human norovirus hyper-mutation revealed by ultra-deep sequencing

Affiliations

Human norovirus hyper-mutation revealed by ultra-deep sequencing

José M Cuevas et al. Infect Genet Evol. 2016 Jul.

Abstract

Human noroviruses (NoVs) are a major cause of gastroenteritis worldwide. It is thought that, similar to other RNA viruses, high mutation rates allow NoVs to evolve fast and to undergo rapid immune escape at the population level. However, the rate and spectrum of spontaneous mutations of human NoVs have not been quantified previously. Here, we analyzed the intra-patient diversity of the NoV capsid by carrying out RT-PCR and ultra-deep sequencing with 100,000-fold coverage of 16 stool samples from symptomatic patients. This revealed the presence of low-frequency sequences carrying large numbers of U-to-C or A-to-G base transitions, suggesting a role for hyper-mutation in NoV diversity. To more directly test for hyper-mutation, we performed transfection assays in which the production of mutations was restricted to a single cell infection cycle. This confirmed the presence of sequences with multiple U-to-C/A-to-G transitions, and suggested that hyper-mutation contributed a large fraction of the total NoV spontaneous mutation rate. The type of changes produced and their sequence context are compatible with ADAR-mediated editing of the viral RNA.

Keywords: Hyper-mutation; Next-generation sequencing; Norovirus; RNA virus.

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Figures

Fig. 1
Fig. 1
NoV genetic map, regions sequenced, and setup of transfection assays. A. In the NoV genetic map, the VP1 capsid gene is shown in red. Molecular clones encompassing the entire VP1 gene were sequenced by the Sanger method. Illumina sequencing was used to analyze smaller regions mapping to the S domain of VP1 and the hyper-variable domain P2 (dark red bars). B. An infectious cDNA clone was transfected in HEK293 cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase, allowing for transcription of plus-strand NoV genomic RNA. A primer annealing to minus-strand copies was used for RT-PCR amplification and sequencing. Colored circles represent mutations/variants.
Fig. 2
Fig. 2
Distribution of U-to-C mutations along a VP1 region in sequences derived from transfected HEK293 cells. The alignment on top shows two examples of highly mutated reads from each transfection assay. The heat map below indicates, for each nucleotide site, the total number of deep-sequencing reads carrying a U-to-C mutation (see color legend).
Fig. 3
Fig. 3
Analysis of hyper-mutation patterns. A. Distribution of the number of U-to-C mutations per deep sequencing read in each of the three transfection assays. The red histograms show the observed counts and the blue line indicates the counts expected from a Poisson model of rare random events. The single parameter of the Poisson distribution was calibrated using the number of reads carrying zero or one mutations. The strong deviation between observed and expected counts shows that sequence reads carrying multiple mutations were more frequent than expected from the Poisson model. Based on this, hyper-mutated reads were defined as those carrying five or more mutations. B. Reproducibility of U-to-C mutation frequency in three transfection assays. In the graphs, each data point corresponds to an U-containing nucleotide site, and the number of times a U-to-C mutation was observed in deep-sequencing reads is plotted for each pair of transfection assays (also represented in the heat map of Fig. 2). From left to right, Spearman correlations were 0.860, 0.855, and 0.955 (p < 10− 12 in all cases). C. Neighbor base preferences for U-to-C and A-to-G hyper-mutation. The histograms show the frequency of U, A, G, and C among 3´neighbors of U-to-C mutations (left), and the frequency of U, A, G, and C among 5´neighbors of A-to-G mutations (right). The crossed lines indicate these same frequencies among non-mutated bases (null expectation). The error bars indicate the SEM frequency from three transfection assays.

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References

    1. Asanaka M., Atmar R.L., Ruvolo V., Crawford S.E., Neill F.H., Estes M.K. Replication and packaging of Norwalk virus RNA in cultured mammalian cells. Proc. Natl. Acad. Sci. U. S. A. 2005;102:10327–10332. - PMC - PubMed
    1. Bodhidatta L., Abente E., Neesanant P., Nakjarung K., Sirichote P., Bunyarakyothin G., Vithayasai N., Mason C.J. Molecular epidemiology and genotype distribution of noroviruses in children in Thailand from 2004 to 2010: a multi-site study. J. Med. Virol. 2015;87:664–674. - PubMed
    1. Bull R.A., White P.A. Mechanisms of GII.4 norovirus evolution. Trends Microbiol. 2011;19:233–240. - PubMed
    1. Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T.L. BLAST +: architecture and applications. BMC Bioinformatics. 2009;10 421–10. - PMC - PubMed
    1. Carlsson B., Lindberg A.M., Rodriguez-Diaz J., Hedlund K.O., Persson B., Svensson L. Quasispecies dynamics and molecular evolution of human norovirus capsid P region during chronic infection. J. Gen. Virol. 2009;90:432–441. - PubMed

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