Near-complete torque teno virus (TTV) genome identified in a blood donor infected with hepatitis B virus (HBV)

ABSTRACT

We have identified a near full-length torque teno virus (TTV) genome sequence in plasma from a blood donor infected with hepatitis B virus. The consensus sequence of TTV was extracted from Nanopore metagenomic sequencing. The identified TTV open reading frame 1 is 3,062 nucleotides (nt) long and shares 90%–100% identity with other human TTVs.

ANNOUNCEMENT

Torque teno virus (TTV) is a single-stranded DNA virus in the Anelloviridae family, which also includes Torque teno mini virus and torque teno midi virus (1–3). These non-enveloped viruses have circular genomes (~2.0–3.9 kilobases) and a conserved untranslated region that regulates replication. The TTV genome encodes three overlapping open reading frames (ORFs), with ORF1 encoding structural proteins, and ORF2 and ORF3 involved in replication and immune evasion (1–3). TTV exists in alpha, beta, and gamma species with 29, 12, and 15 genotypes (4–6). TTV is ubiquitous in humans, including blood donors, and is typically non-pathogenic (3, 7, 8). However, its replication increases in immunocompromised individuals (9, 10) or those with hepatitis, suggesting potential immune system interactions in liver disease (11–13).

To evaluate potential transfusion-transmitted infections and blood safety, we analyzed four de-identified hepatitis B-infected plasma samples obtained from the American Red Cross Blood Bank, Gaithersburg, MD, USA, using Nanopore metagenomic sequencing. Viral DNA/RNA was extracted from 200 µL of plasma with the Zymo Viral DNA/RNA Kit, followed by unbiased amplification using the Cytiva rolling circle amplification (RCA) kit protocol to generate high yields of viral single-stranded DNA, ideal for Nanopore sequencing (14). Once the RCA was complete, amplified nucleic acids were purified using the GeneJET PCR Purification Kit to remove any remaining contaminants such as enzymes or primers. Sequencing libraries were prepared with the nanopore native barcoding kit 24 (V14) protocol, including a spike-in lambda control, and loaded onto an R10 flow cell for Nanopore MinION sequencing. Data collection and basecalling were performed with Nanopore MinKNOW (version 22.12.7) and Guppy (version 6.4.6) using default presets. Taxonomy analysis was conducted using the EDGE Bioinformatics web tool with default parameters (15), and reference mapping was performed with CLC Genomic Workbench (version 23.0.5) using Minimap2.

The sequencing reads were first filtered out by reference mapping against the human genome reference (hg38, GenBank ID: GCF_000001405.40) and the lambda genome reference (NC_001416). Taxonomy results revealed that the most abundant species are HBV (210 hits) and Alphatorquevirus (46 hits), while the remaining reads were mapped to other non-human microbial genomes. Subsequent target reference analysis showed that out of 6,279 total reads, 67 mapped to TTV (GenBank ID: MN765343) and 120 to HBV (GenBank ID: GQ475311). The identified TTV exhibited 98% genome coverage with a depth of coverage of 33.4× and a GC content of 49.3%, resulting in a nearly complete viral genome sequence. A 3,062 bp consensus sequence was extracted using the CLC consensus extraction tool for evolutionary analysis. The identified TTV and near full-length TTV genotypes from the NCBI database were aligned using ClustalW in MEGA version 11 for phylogenetic analysis (16). The Neighbor-Joining phylogenetic tree (Fig. 1) revealed that the sequence BB02_W200314045308P (accession No. PQ438050) exhibited 100% nucleotide similarity with the TTV-3 genotype (GenBank ID: MN765343). Additionally, PQ438050 encodes a 731 amino acid polyprotein ORF1. An NCBI BLASTp search showed 93.8% similarity with an Alphatorquevirus homin3 (GenBank ID: XBU06458).

Fig 1.

Phylogenetic tree of TTV3 ORF1 was constructed using the Neighbor-Joining method, including representative genotypes from the TTV genus. Sequences identified in this study are marked with a black circle. Bootstrap analysis with 500 replicates was conducted, and the resulting support percentages are displayed at the branches, reflecting the confidence level for each genotype.

DATA AVAILABILITY

The newly identified TTV-3 sequence has been deposited in GenBank under the accession no. PQ438050. Mapped raw reads have been deposited in the SRA database under SRR29828227.

ETHICS APPROVAL

This study was determined to be exempt by the Food and Drug Administration’s Research Involving Human Subjects Committee under 45 CFR 46.101(b)4, Category 4, as the research involved the study of existing specimens where the subjects cannot be identified, directly or through identifiers linked to the subjects. The research study does not require FDA IRB review and approval because it is exempt from the requirements of 45 CFR Part 46.