Human norovirus hyper-mutation revealed by ultra-deep sequencing

Abstract

Human norovirus hyper-mutation revealed by ultra-deep sequencing, (2016), doi: 10.1016/j.meegid.2016.04.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 2 AbstractHuman 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 has not been quantified previously. Here, we analysed the intrapatient 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.Graphical abstract: this optional item will be prepared and submitted upon manuscript acceptance.

Characterization of intra- and inter-host norovirus P2 genetic variability in linked individuals by amplicon sequencing

“…Of note, 96.5% of these substitutions were transitions (58.1% of the transitions were substitutions U-to-C, 19.4% were A-to-G), and only 3.5% were transversions (G-to-U) ( Table 5 ). Since HuNoV mutants with a high proportion of U-to-C and A-to-G transitions have been described in clinical samples and double-stranded RNA-specific adenosine deaminases (ADAR)-mediated editing of the viral RNA was suggested as the molecular mechanism responsible for them [ 26 ], we analyzed their sequence context. Although the chi-square test did no show significant differences compared to the percentages found for conserved sites, of all U-to-C transitions, the 3’ neighbor nucleotide was U in 40% of cases, A in 26.7%, G in 20% and C in 13.3%, which tend to the sequence context compatible with ADAR-mediated editing of the viral RNA.…”

Static and Evolving Norovirus Genotypes: Implications for Epidemiology and Immunity

“…The strategy of amplification was similar to that published by Eden et al [ 67 ] for GII.4 viruses, and our method was robust for a number of GII noroviruses (GII.1, GII.2, GII.3, GII.4, GII.6, GII.12, and GII.17), and from samples stored for over 40 years [ 35 ]. Several groups have explored the intra-host diversity of noroviruses by NGS using partial regions of the genome [ 23 , 68 ]; however, our approach extended these findings by allowing high-resolution analysis at every nt position in the coding sequence of the genome. We first examined the intra-host evolution of GII.4, GII.6 and GII.17 noroviruses within a single patient, and observed that only the GII.4 viruses presented a gradual increase in the number of mutations, which in some cases resulted in aa substitutions in areas regarded as important antigenic sites.…”

Mechanisms of viral mutation

“…ADARs also exhibit sequence context preferences, although less marked than in the case of APOBECs [ 68 ]. ADAR-driven hyper-mutation was first demonstrated in measles virus [ 69 ] and has since been suggested for a variety of RNA viruses including human parainfluenza virus [ 70 ], respiratory syncytial virus [ 71 ], lymphocytic choriomeningitis virus [ 72 ], Rift Valley fever virus [ 73 ], and noroviruses [ 74 ].…”