Dynamics of the Stool Virome in Very Early-Onset Inflammatory Bowel Disease

Abstract

Background and aims: Dysbiosis of the gut microbiota is a well-known correlate of the pathogenesis of inflammatory bowel disease [IBD]. However, few studies have examined the microbiome in very early-onset [VEO] IBD, which is defined as onset of IBD before 6 years of age. Here we focus on the viral portion of the microbiome-the virome-to assess possible viral associations with disease processes, reasoning that any viruses potentially associated with IBD might grow more robustly in younger subjects, and so be more detectable.

Methods: Virus-like particles [VLPs] were purified from stool samples collected from patients with VEO-IBD [n = 54] and healthy controls [n = 23], and characterized by DNA and RNA sequencing and VLP particle counts.

Results: The total number of VLPs was not significantly different between VEO-IBD and healthy controls. For bacterial viruses, the VEO-IBD subjects were found to have a higher ratio of Caudovirales vs to Microviridae compared to healthy controls. An increase in Caudovirales was also associated with immunosuppressive therapy. For viruses infecting human cells, Anelloviridae showed higher prevalence in VEO-IBD compared to healthy controls. Within the VEO-IBD group, higher levels of Anelloviridae DNA were also positively associated with immunosuppressive treatment. To search for new viruses, short sequences enriched in VEO-IBD samples were identified, and some could be validated in an independent cohort, although none was clearly viral; this provides sequence tags to interrogate in future studies.

Conclusions: These data thus document perturbations to normal viral populations associated with VEO-IBD, and provide a biomarker-Anelloviridae DNA levels-potentially useful for reporting the effectiveness of immunosuppression.

Keywords: VEO-IBD; Very early-onset inflammatory bowel disease; metagenome; microbiome; virome.

Figure 1.

Overview of cohort characteristics and data analysis.

Figure 2.

Comparing bacteriophage populations in samples from VEO-IBD patients vs healthy controls. [A] Boxplot of the relative abundance of different viral taxa at the family level. [B] Correlation between the abundance of Caudovirales and Microviridae. The p- and r-values were calculated by Spearman’s correlation test. Linear regression with 95% confidence interval is shown. [C] Comparison between the abundance of Caudovirales and Microviridae in VEO-IBD patients [n = 54] and healthy controls [n = 23]. [D] Comparison between the abundance of Caudovirales and Microviridae in active VEO-IBD patients [n = 20] and inactive VEO-IBD patients [n = 16]. [E] Comparison between the abundance of Caudovirales and Microviridae in VEO-IBD patients with immunosuppressive treatment [n = 24] and without immunosuppressive treatment [n = 30]. The Wilcoxon rank-sum test was used throughout to test for differences.

Figure 3.

Viral taxa that are differentially present in VEO-IBD vs healthy controls. The y-axis at the top presents reads per million total mapped reads [RPM] of each taxon with log₁₀ transformation. The x-axis at the left presents each different taxon. [A] Viral taxa differed among healthy controls, and inactive and active patients. [B] Viral taxa differed among healthy controls, and immunosuppressive untreated and treated patients.

Figure 4.

Anelloviridae prevalence. [A] Comparison between the prevalence of Anelloviridae in VEO-IBD patients [n = 54] and healthy controls [n = 23]. The y-axis indicates the percentage of positive subjects with Anelloviridae. Positive calls required that at least one Anelloviridae genome was more than 33% covered by sequence reads. Samples were compared using Fisher’s exact test. [B] Comparison between the prevalence of Anelloviridae in VEO-IBD patients with immunosuppressive treatment [n = 24] and VEO-IBD patients without immunosuppressive treatment [n = 30]. The y-axis is as in A. Samples were compared using Fisher’s exact test.

Figure 5.

K-mer-based analysis. [A] Diagram of the analytical approach. [B] Presence/absence heatmap of differentially present RNA 31-mers between VEO-IBD and healthy controls. Red-coloured cells indicate presence while white-coloured cells indicate absence. The y-axis indicates different 31-mers [n = 492], and the y-axis coloir bar represents the nucleotide database annotation of 31-mers. [C] As in B, but showing differentially present 31-mers between IBD and healthy controls from the iHMP dataset.