The gut of healthy newborns is usually devoid of viruses at birth, but it is rapidly colonized afterwards [28, 31]. Still relatively few studies have focused on the assembly of the gut virome within the first year of life and the factors that influence it [6, 27, 28, 31] and even less is known about the environmental exposures that shape the gut virome.
Here, we leveraged a massive gut virome dataset from healthy infants at 1-year of age, and integrated measures of viral diversity such as sequence composition, viral hosts, and phage lifestyles [9], (see Fig. 1) with social, pre-, peri- and postnatal environmental exposures. We revealed the effects of these exposures on viral community and the possible effects on metabolism.
In previous reports, Crassvirales (class Caudoviricetes) and Microviridae (class Malgrandaviricetes) phages were found to be the two most abundant viral groups in the adult human gut, with their relative abundance being negatively correlated [19, 47–50]. Here, in one-year-old infants, a similar observation was made, members of the Caudoviricetes and Malgrandaviricetes classes were the most abundant phages.
Interestingly, ongoing exposures such as having older siblings and residential location, as well as past exposures (e.g., birth weight, preeclampsia) were linked with gut virome composition at one year of age. However, it is still possible that the prenatal and perinatal exposures still influenced the immune education earlier in life and remnants of the interplay are still tangible at 1-year of age [5]. Among the exposures significantly influencing the gut virome composition, the largest effect sizes were from residential location (rural vs. urban) and having older siblings (see Fig. 2C and 2D). Interestingly, urbanization has been reported to have a significant impact on the composition of the adult viral community, with individuals living in urban areas having higher abundance of Lactococcus (family Streptococcaceae) phages [51]. The latter is presumably associated with the consumption of dairy products. We show that the living environment also affects the gut virome of infants, and that Streptococcaceae targeting phages are also more abundant in infants living in urban areas, possibly reflecting differences in dietary habits rather than residence per se (Fig. 3).
Having older siblings influences the development of the bacterial community in early life [34, 52, 53] and here we show that having older siblings is also associated with gut virome composition at one year of age. Importantly, focussing on the curated DNA phage community (i.e. not the total vOTU pool, but only the fraction predicted as being bacteriophages) we recently demonstrated that phageome imbalances are associated with increased risk of developing before school age, but also that having older siblings was negatively associated with higher virome asthma signature score, implying a lower asthma rate, showing how the influence of environmental exposures on virome composition also have implications for child health [22]. Importantly, from a translational angle, early-life exposures may affect the establishment of health phenotypes, such as the protective role of breastfeeding against eukaryotic-viral infections in the neonatal period [31]. Combining gut bacterial compositional data with gut virome composition (Figure S3A and S5B-C) in our cohort elucidates the co-abundance of phages and their hosts, underlying the role of phage-host interactions in shaping the GM. Most of these viruses (Figure S3A) have temperate lifestyles, as evidenced by the presence of genes coding for integrases. Thus, these temperate phages appear to have the ability to integrate their genome into the bacterial hosts and become prophages at some point.
Gut virome members have the potential to modulate biochemical processes [12, 13, 54]. The functional prediction of the genes derived from vOTUs co-varying with exposures, revealed up to 90% of genes with unknown functions. It emphasizes that proteins with yet uncharacterized functions are potentially playing a role in the regulation of human host phenotypes. Certain predicted gene functions linked to metabolic activities, such as alanine, aspartate and glutamate metabolism, amino sugar and nucleotide sugar metabolism and glycolysis/gluconeogenesis, which are likely associated with dietary intake and degradation of macronutrients, were associated with fish in the diet, birth weight, residence location and egg in the diet (Fig. 4A). Birth weight also show negative correlation with higher virome asthma signature score (asthma rate) [22], and time-to-asthma analysis proved that the effects of phages on asthma were additive and statistically independent of the bacterial gut microbiome component [22]. Maternal obesity alters fatty acid metabolism and changes in gene expression of lipid metabolism in infants, which cause a higher risk of developing obesity and its complications, neuropsychiatric disorders and asthma [55, 56]. We find here that viral genes associated with normal weight mothers were predominantly enriched in fatty acid biosynthesis compared to obese mothers, which may be an intermediate pathway by which maternal obesity affects child health. In addition, for biotin metabolism, which is known to be impaired by severe obesity [57], many phage genes are also observed to be enriched in infants from mothers with BMI below 25 in our data. The mothers enrolled in the cohort participated in a randomized clinical trial where they were randomized to receiving fish oil or a placebo from week 24 of pregnancy to one week after birth [41, 58]. The design also examined the difference in vitamin D between high and standard doses [42], which had no effect on the viral community in our analysis. Of note, the supplementation of fish oil during pregnancy was not found to influence the gut bacterial component at age one year. Here we report that the same intervention has some influence on the gut virome at age one year, but the effect is only borderline significant. The infants of mothers that received fish oil had viral genes involved in lysine biosynthesis, glycerophospholipid metabolism, and purine metabolism – metabolic activities that have been associated to fish oil supplementation [59, 60], but never attributed to gut virome composition. Interestingly, most of these metabolism-related genes were conserved across temperate vOTUs targeting Ruminococcus, Faecalibacterium and Prevotella spp. (Fig. 4B). These genera have been consistently reported to be enriched in Danish and American subjects with a diet rich in carbohydrates, resistant starch, and fibers, and being determinants of the so-called Prevotella-enterotype [61, 62]. The Prevotella-enterotype is established early in life (between 9–36 months of age) [63–65] and have been previously suggested as markers of GM maturity at age one year [2, 66, 67]. Stokholm et al. (2018) reported delayed GM maturation as a risk factor for later development of asthma indicating the importance of these microbes for immune maturation.
Although our study is currently unable to assess how these gut virome associated genes are actively involved in either enhancing either phage or host fitness, or both, our data as well as our recent finding, that early life gut virome imbalance is associated with increased asthma before school age risk [22] underlines the potential importance of bacteriophage-encoded metabolic genes and delivers an initial insight of the type of metabolic content conveyed by the gut virome in association to environmental variables.
In summary, our data provides detailed insight into the influence of common environmental factors that shape the gut virome during early life. We also uncover that key gut metabolic functions can be encoded by viral genes, which suggest that, in addition of shaping gut bacteriome composition, phages may directly play a role in metabolic activities.