CrAssphages are human gut bacteriophages with potential use as an indicator of human fecal contamination in water and other environmental systems. We determined the prevalence and abundance of crAssphages in water, food, and fecal samples and compared these estimates with the prevalence of norovirus. Samples were tested using two crAssphage-specific qPCR assays (CPQ056 and TN201-203) and for norovirus using TaqMan realtime RT-PCR. CrAssphage was detected in 40% of human fecal specimens, 61% of irrigation water samples, 58.5% of stream water samples, and 68.5% of fresh leafy greens samples. Interestingly, across all sample categories, crAssphage concentrations were 2-3 log10 higher than norovirus concentrations. The correlation of detection of crAssphage and norovirus was significant for the irrigation water samples (r = 0.74, p = 7.4e-06). Sequences obtained from crAssphage positive samples from human fecal and stream water samples phylogenetically clustered with genotype I crAssphages, whereas sequences derived from irrigation water samples clustered differently from other genotypes. Our data show that crAssphages were prevalent in norovirus-positive water samples and in fresh leafy green samples, there was a strong correlation between the presence of crAssphage and norovirus. CrAssphage genomic copies were consistently higher than norovirus copies in all sample types. Overall, our findings suggest that crAssphages could be used as reliable indicators to monitor fecal-borne virus contamination within the food safety chain.

Noroviruses are associated with one fifth of diarrheal illnesses globally and are not yet preventable with vaccines. Little is known about the effects of norovirus infection on infant gut microbiome health, which has a demonstrated role in protecting hosts from pathogens and a possible role in oral vaccine performance. In this study, we characterized infant gut microbiome changes occurring with norovirus-associated acute gastroenteritis (AGE) and the extent of recovery. Metage-nomic sequencing was performed on the stools of five infants participating in a longitudinal birth cohort study conducted in León, Nicaragua. Taxonomic and functional diversities of gut micro-biomes were profiled at time points before, during, and after norovirus infection. Initially, the gut microbiomes resembled those of breastfeeding infants, rich in probiotic species. When disturbed by AGE, Gammaproteobacteria dominated, particularly Pseudomonas species. Alpha diversity in-creased but the genes involved in carbohydrate metabolism and glycan biosynthesis decreased. After the symptoms subsided, the gut microbiomes rebounded with their taxonomic and functional communities resembling those of the pre-infection microbiomes. In this study, during disruptive norovirus-associated AGE, the gut microbiome was temporarily altered, returning to a pre-infection composition a median of 58 days later. Our study provides new insights for developing probiotic treatments and furthering our understanding of the role that episodes of AGE have in shaping the infant gut microbiome, their long-term outcomes, and implications for oral vaccine effectiveness. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.