Development of PCR assays with nested primers specific for differential detection of three human anelloviruses and early acquisition of dual or triple infection during infancy.

We recently identified a novel human virus classifiable into a third group in the genus Anellovirus, tentatively designated torque teno midi virus (TTMDV), with a circular DNA genome of 3.2 kb and genomic organization resembling those of torque teno virus (TTV) (3.8 to 3.9 kb) and torque teno mini virus (TTMV) (2.8 to 2.9 kb). TTMDV was characterized by extreme genetic diversity similar to the TTV and TTMV genomes. Taking advantage of universal and virus species-specific primers derived from a highly conserved area located just downstream of the TATA box of the TTV, TTMDV, and TTMV genomes, a PCR method with simultaneous amplification of the genomic DNAs of these three anelloviruses in the first round and subsequent differential amplifications of these viruses in the second round was developed. High prevalence of TTMDV viremia was seen in adults (75/100 [75%]), comparable with the prevalences of TTV viremia (100%) and TTMV viremia (82%). Although none of 10 cord blood samples had detectable TTV, TTMDV, and TTMV DNAs, the prevalences of these three anelloviruses increased with the number of months after birth of the individual and reached 100% for individuals at one year of age. Dual or triple infection of TTV, TTMDV, and/or TTMV was seen in 10 (47.6%) of 21 infants 9 to 180 days of age and more frequently among infants 181 to 364 days of age (20/23 [86.9%]), comparable with the 93.1% (243/261) prevalence among subjects 1 to 81 years of age, indicating early acquisition of dual or triple anellovirus infection during infancy.