Anne L. Timmerman

Background: Among the most abundant viruses in the human blood virome are anelloviruses, including alpha ‐, beta ‐, and gammatorqueviruses (TTV, TTMV, and TTMDV). Whether anelloviruses are transmitted through kidney transplantation at a young age and subsequently persist in their new host is unknown. This study investigates the dynamics and composition of the anellome in the blood of six pediatric donor–recipient pairs, with monitoring beginning before transplantation and continuing until 2 years after transplantation. Methods: Donors were sampled once, before donation, while recipients were sampled before and multiple times after transplantation (median 6.5 samples). Quantitative PCR, rolling circle amplification, Illumina sequencing, and SCANellome V2 analysis were used to detect, characterize, and compare anellovirus presence in donors and recipients. Results: At baseline, four out of six donors tested positive for TTV by quantitative anellovirus PCR, three of whom had sufficient viral loads to enable genomic comparison with their recipients. All recipients tested positive at baseline, five had a moderate viral load (< 10 ⁵ copies/mL), and one had > 10 ⁹ copies of TTV DNA/mL. This recipient was already immunosuppressed before transplantation, and only in this recipient was a donor‐derived anellovirus identified. This lineage was detected among 15 other lineages in the recipient and matched one of the four lineages identified in the donor. Conclusion: This finding demonstrates that anellovirus transmission from donor to recipient occurs in pediatric kidney transplantation. This may be associated with pre‐existing immunosuppression.

Anelloviruses are ubiquitous, diverse viruses in the human virome not causing disease. In adults, the anellovirus composition (anellome) is dominated by alphatorqueviruses. However, infants seem to acquire their first anellovirus infections beginning with beta - and gammatorquevirus, soon followed by alphatorquevirus. Transmission via human milk is suggested as a potential transmission route. Consequently, we hypothesize that milk may contain predominantly beta- and gammatorqueviruses. That would also imply that compartmentalization between milk and serum takes place as the anellome in adults is dominated by alphatorqueviruses. We therefore examined anellovirus distribution in paired serum and milk samples of lactating mothers in the first 15 months after delivery ( n = 30). Unexpectedly, genus-specific qPCRs revealed that the majority of serum and milk were negative for anelloviruses 2–6 months postpartum (4 serum and 1 milk positives out of 13), whereas positive from 6 months postpartum onwards (11 serum and 8 milk positives out of 16). Importantly, a dominance of beta- and gammatorquevirus in both serum and milk from 6 months postpartum was found. We also found significantly higher anellovirus loads in serum compared to milk, with no obvious difference in anellovirus genera presence between compartments. Anellovirus variant analysis using Oxford Nanopore sequencing revealed that 31 out of 45 variants were shared with the corresponding serum of the mother. We conclude that anellovirus compartmentalization between the circulation and the mammary glands could not be detected. IMPORTANCE: Anelloviruses are omnipresent in the human population, yet their transmission routes, especially in early life, remain unclear. This study focused on compartmentalization between blood and the mammary gland to investigate the likelihood of virus transmission via milk. We show that serum and milk samples generally share anellovirus genera and lineages. Interestingly, serum as well as milk samples collected between 2 and 6 months postpartum tested mainly negative for anelloviruses, but those received thereafter were dominated by beta- and gammatorquevirus, reflecting the initial anellovirus colonizers observed in children.

One continuous companion and one of the major players in the human blood virome are members of the Anelloviridae family. Anelloviruses are probably found in all humans, infection occurs early in life and the composition (anellome) is thought to remain stable and personal during adulthood. The stable anellome implies a great balance between the host immune system and the virus. However, the lack of a robust culturing system hampers direct investigation of interactions between virus and host cells. Other techniques, however, including next generation sequencing, AnelloScan-antibody tests, evolution selection pressure analysis, and virus protein structures, do provide new insights into the interactions between anelloviruses and the host immune system. This review aims at providing an overview of the current knowledge on the immune mechanisms acting on anelloviruses and the countering viral mechanisms allowing immune evasion.

Members of the Anelloviridae family dominate the blood virome, emerging early in life. The anellome, representing the variety of anelloviruses within an individual, stabilizes by adulthood. Despite their supposedly commensal nature, elevated anellovirus concentrations under immunosuppressive treatment indicate an equilibrium controlled by immunity. Here, we investigated whether anelloviruses are sensitive to the immune activation that accompanies a secondary infection. As a model, we investigated 19 health care workers (HCWs) with initial SARS-CoV-2 infection, with blood sampling performed pre and post infection every 4 weeks in a 3-month-follow-up during the early 2020 COVID-19 pandemic. A concurrently followed control group (n = 27) remained SARS-CoV-2-negative. Serum anellovirus loads were measured using qPCR. A significant decrease in anellovirus load was found in the first weeks after SARS-CoV-2 infection, whereas anellovirus concentrations remained stable in the uninfected control group. A restored anellovirus load was seen approximately 10 weeks after SARS-CoV-2 infection. For five subjects, an in-time anellome analysis via Illumina sequencing could be performed. In three of the five HCWs, the anellome visibly changed during SARS-CoV-2 infection and returned to baseline in two of these cases. In conclusion, anellovirus loads in blood can temporarily decrease upon an acute secondary infection.

Human anelloviruses (AVs) are extremely genetically diverse, widespread in the human population, and cause chronic infections. However, the evolutionary dynamics of AVs within single hosts is currently unknown, and it is unclear whether these changes have an implication on the long-term persistence of AVs in the host. Here, we assessed the evolutionary dynamics of six AV lineages during 30 years of chronic infection at single host resolution. The total number of substitutions and the number of variable sites increased over time. However, not all substitutions reached population fixation, showing that AV lineages form heterogeneous swarms within the host. Most substitutions occurred within a hypervariable region (HVR) located between nucleotide positions 800 and 1300 of ORF1, which is known to be located within the spike domain. Different regions of the ORF1 gene undergo either positive or negative selection pressure. Sites under strong diversifying selection pressure were detected in the HVR, while the majority of the sites under purifying selection were detected outside this region. The HVR may play the role of an immunological decoy that prevents antibodies from binding to more vulnerable parts of ORF1. Moreover, the frequent substitutions in this region may increase the chances of AV particles escaping immune recognition.

Despite significant attention on anellovirus research, the interaction between the anellovirus virome and the human host remains unknown. We show the dynamics of APOBEC3-mediated cytidine deaminase activity on anelloviruses during a 30-year period of chronic infection and postulate that this antiviral mechanism controls anelloviruses. These results expand our knowledge of anellovirus-host interactions, which may be important for the design of gene therapies.

Knowledge of the persistence of AVs in humans is crucial to our understanding of the nature of AV infection (chronic or acute) and the role of AV in the host. We therefore investigated the dynamics of anellovirus infection in two healthy people followed up for 30 years. Our findings suggest that the human blood anellovirus virome (anellome) remains stable and personal for decades.

Anelloviruses (AVs) are found in the vast majority of the human population and are most probably part of a healthy virome. These viruses infect humans in the early stage of life, however, the characteristics of the first colonizing AVs are still unknown. We screened a collection of 107 blood samples from children between 0.4 and 64.8 months of age for the presence of three AV genera: the Alpha-, Beta- and Gammatorquevirus. The youngest child that was positive for AV was 1.2 months old, and a peak in prevalence (100% of samples positive) was reached between the twelfth and eighteenth months of life. Intriguingly, the beta- and gammatorqueviruses were detected most at the early stage of life (up to 12 months), whereas alphatorqueviruses, the most common AVs in adults, increased in prevalence in children older than 12 months. To determine whether that order of colonization may be related to oral transmission and unequal presence of AV genera in breast milk, we examined 63 breast milk samples. Thirty-two percent of the breast milk samples were positive in a qPCR detecting beta- and gammatorqueviruses, while alphatorqueviruses were detected in 10% of the samples, and this difference was significant (p = 0.00654). In conclusion, we show that beta- and gammatorqueviruses colonize humans in the first months of life and that breastfeeding could play a role in AV transmission.