ABSTRACT
Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.
ABSTRACT
Although UV-induced mutagenesis has been studied extensively, the precise mechanisms that convert UV-induced DNA damage into mutations remain elusive. One well-studied mechanism involves DNA polymerase (Pol) η and ζ, which produces C > T transitions during translesion synthesis (TLS) across pyrimidine dimers. We previously proposed another biochemical mechanism that involves multiple UV-irradiations with incubation in the dark in between. The incubation facilitates spontaneous deamination of cytosine in a pyrimidine dimer, and the subsequent UV irradiation induces photolyase-independent (direct) photoreversal that converts cytosine into monomeric uracil residue. In this paper, we first demonstrate that natural sunlight can induce both mutational processes in vitro. The direct photoreversal was also reproduced by monochromatic UVB at 300 nm. We also demonstrate that post-irradiation incubation in the dark is required for both mutational processes, suggesting that cytosine deamination is required for both the Pol η/ζ-dependent and the photoreversal-dependent mechanisms. Another Y-family polymerase Pol ι also mediated a mutagenic TLS on UV-damaged templates when combined with Pol ζ. The Pol ι-dependent mutations were largely independent of post-irradiation incubation, indicating that cytosine deamination was not essential for this mutational process. Sunlight-exposure also induced C > A transversions which were likely caused by oxidation of guanine residues. Finally, we constructed in vitro mutation spectra in a comparable format to cancer mutation signatures. While both Pol η-dependent and photoreversal-dependent spectra showed high similarities to a cancer signature (SBS7a), Pol ι-dependent mutation spectrum has distinct T > A/C substitutions, which are found in another cancer signature (SBS7d). The Pol ι-dependent T > A/C substitutions were resistant to T4 pyrimidine dimer glycosylase-treatment, suggesting that this mutational process is independent of cis-syn pyrimidine dimers. An updated model about multiple mechanisms of UV-induced mutagenesis is discussed.
