Expansion of Betatorquevirus and/or Gammatorquevirus in Patients with Severe Clinical Outcomes of the Liver Diseases

Abstract

Anellovirus (AV) is a ubiquitous virus in the human population. Individuals can be infected with multiple AV genera and species to form a heterogeneous repertoire, termed the anellome. Using advanced methods, we examined the anellomes from 12 paired serum and liver samples, as well as 2701 subjects with different clinical diagnoses. Overall, anellomes are remarkably individualized, with significant among-group differences (Kruskal-Wallis test p = 6.6 × 10^-162 for richness and p = 7.48 × 10^-162 for Shannon entropy). High dissimilarity scores (beta diversity) were observed between patient groups, except for paired serum and liver samples. At the population level, the relative abundance of combinational AV genus Betatorquevirus (torque teno mini viruses, TTMV), and Gammatorquevirus (torque teno midi viruses, TTMDV) exhibited an exponential distribution with a low bound point at 32%. Defined by this value, the AV TTMV/TTMDV-expanded anellome was significantly enriched among patients with acute liver failure (31.7%) and liver transplantation (40.7%), compared with other patient groups (χ² test: p = 4.1 × 10^-8-3.2 × 10^-3). Therefore, anellome heterogeneity may be predictive of clinical outcomes in certain diseases, such as liver disease. The consistency of anellome between paired serum and liver samples indicates that a liquid biopsy approach would be suitable for longitudinal studies to clarify the causality of the AV TTMV/TTMDV-expanded anellome in the outcomes of liver disease.

Keywords: anellovirus; hepatitis C virus; hepatocellular carcinoma; next-generation sequencing; virome.

Figure 1

Anellovirus profiles of paired serum and liver tissue samples. Twelve paired serum and liver samples were compared with regard to the anellome composition resolved at the level of AV species (A) or genus (B). Anellome richness (C), Shannon entropy (D), and anellovirus titer (E) were also compared between serum and liver samples.

Figure 2

Results of principal coordinate analysis of anellomes among ten groups. The analysis was conducted using either Bray–Curtis (A) or Yue–Clayton (B) dissimilarity matrices. The clinical diagnosis of each patient group is indicated. DLS, dengue-like symptom.

Figure 3

Serum/plasma anellomes of subjects in relation to patient groups. Anellome compositions, with the determination of relative abundance, were summarized at the level of anellovirus species (A) and genus (B). Groups G1–8 stand for acute liver failure, liver transplantation, kidney transplantation, lung transplantation, blood donors, blood donors from Spain, dengue-like symptoms, and febrile children, respectively.

Figure 4

Box and whisker plots representing anellome diversity. The number of anellovirus species (A) and Shannon entropy (B) among the eight groups are represented by boxes (second quartile, median and third quartile diversity) and whiskers (minimum and maximum diversity). Dots indicate outliers. Among-group comparisons were carried out using the Kruskal–Wallis test. Groups G1–8 stand for acute liver failure, liver transplantation, kidney transplantation, lung transplantation, blood donors, blood donors from Spain, dengue-like symptoms, and febrile children, respectively.

Figure 5

Histogram showing the relative abundance of combinational TTMV and TTMDV in the anellomes from 530 serum/plasma samples. The data distribution fit an exponential distribution, in which most samples (78.7%) had TTMV and TTMDV shrunk within the anellome. The χ_min was calculated to be 32, which put 113 samples (21.3%) in the decay region (shaded area).

Figure 6

Prevalence of TTMV/TTMDV-expanded anellomes among different patient groups. The proportion of samples with (orange) and without (blue) TTMV/TTMDV-expanded anellomes was presented for each group. Asterisk indicated the p values of the between-group comparisons (*, p < 0.5; **, p < 0.1; ****, p < 0.001). The group G8 is represented using the right y-axis. Groups G1–8 stand for acute liver failure, liver transplantation, kidney transplantation, lung transplantation, blood donors, and blood donors from Spain, dengue-like symptoms, and febrile children, respectively.

Figure 7

Phylogenetic representation of anellovirus evolutionary dynamics in liver transplantation. Trees were constructed using anellovirus ORF1 sequences of two selected cases (#12 and #23). Time points are indicated using different colors; unlabeled branches show anellovirus reference sequences. AV sequences belonging to the same AV species, exemplified using Torque teno virus 3 (TTV3) in case #12 and Torque teno virus 21 (TTV21) in case #23, were detected at multiple time points before and after liver transplantation. Bootstrap values are indicated on major branches.