New Phylogenetic Groups of Torque Teno Virus Identified in Eastern Taiwan Indigenes

Abstract

Torque teno virus (TTV) is a single-stranded DNA virus highly prevalent in the world. It has been detected in eastern Taiwan indigenes with a low prevalence of 11% by using N22 region of which known to underestimate TTV prevalence excessively. In order to clarify their realistic epidemiology, we re-analyzed TTV prevalence with UTR region. One hundred and forty serum samples from eastern Taiwanese indigenous population were collected and TTV DNA was detected in 133 (95%) samples. Direct sequencing revealed an extensive mix-infection of different TTV strains within the infected individual. Entire TTV open reading frame 1 was amplified and cloned from a TTV positive individual to distinguish mix-infected strains. Phylogenetic analysis showed eleven isolates were clustered into a monophyletic group that is distinct from all known groups. In addition, another our isolate was clustered with recently described Hebei-1 strain and formed an independent clade. Based on the distribution pattern of pairwise distances, both new clusters were placed at phylogenetic group level, designed as the 6th and 7th phylogenetic group. In present study, we showed a very high prevalence of TTV infection in eastern Taiwan indigenes and indentified new phylogenetic groups from the infected individual. Both intra- and inter-phylogenetic group mix-infections can be found from one healthy person. Our study has further broadened the field of human TTVs and proposed a robust criterion for classification of the major TTV phylogenetic groups.

Fig 1. Unrooted Bayesian inferred maximum clade credibility tree of TTV ORF1 well aligned region.

Posterior probabilities are labeled at major branches. Branches with a posterior probabilities equal or above 0.95 are considered as strong supported. Isolates from this study are labeled in bold. The scale bar at the left bottom indicates evolutionary distance in unit of substitutions per site per generation. The semicircular symbols indicate seven major TTV phylogenetic groups. The dash lines indicate tentatively classifications. Tentatively classified strains are labeled for reference. The SANBAN, SAa-01, and SENVG are arbitrarily chosen as representative for showing subgroup 3a, 3b, and 3c respectively. See S1 Fig and S1 Appendix for detail taxa list. *Label TW53A25 including 8 isolates (TW53A25, TW53A27, TW53A28, TW53A29, TW53A32, TW53A34, TW53A35, and TW53A39). **Label TW53A26 including 3 isolates (TW53A26, TW53A30, and TW53A31).

Fig 2. Frequency distribution plot of p-distance for TTVs.

The uncorrected distance matrix is calculated from the same data set as that used for phylogenetic analysis. Y axis represents the frequency counts in each bin. X axis represents the value of uncorrected pairwise distance. Horizontal bars indicate the distribution ranges. The gray zone indicates the overlapping regions between the distribution of intragroup and intergroup distances. Rectangles indicate the distance distribution ranges of each group and divided into left and right regions. Left regions show the distribution range of which pairwise distances are calculated within each group (intragroup distances). Right regions show the distribution range of pairwise distances that compared to strains of other groups (intergroup distances). The slash lines indicate the overlapping gray area of each group. Rectangles of group 6 and group 7 have small left regions because only limited strains are available. The subgroups of group 3 (3a, 3b, and 3c) are indicated by dashed rectangles. Dashed rectangles are also divided into two parts as well as major groups, except the right regions showing the distribution range of pairwise distances that compared to strains from two other subgroups in group 3.

Fig 3. Putative genomic organization of TTV group 6 by using TW53A26 as representative isolate.

ORFs are marked with black arrows. Predicted donor and acceptor sites are linked by dashed border. The dotted line indicated UTR that was not included in our ORF amplicon. ORF1 encoded for 736 amino acids (nt 445–2652), ORF2 for 159 amino acids (nt 152–628), ORF3 for 347 amino acids (nt 152–627 and 2155–2719), ORF4 for 160 amino acids (nt 152–627 and 2357–2360). The numbering of nt 1 (the first) and the nt 3211 (last nucleotide) of TW53A26 is according to nt 225 and nt 3336 of TA278, respectively. Numbers in bracket indicated the corresponding nucleotide position in TA278. The full length of TTV group 6 is about 3,932 nt calculated by assuming the UTR is conserved with Hel32, a strain with the same length of translated ORF1. *Since it is not included in consensus sequence, this group that a nonsense mutation of ORF1 at nt 523 of isolate TW53A26 is ignored here.