Rolling-circle amplification of Torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup

Abstract

Multiply primed rolling-circle amplification is a novel technology that uses bacteriophage phi29 DNA polymerase to amplify circular DNA molecules, without the need for prior knowledge of their sequences. In an attempt to detect Torque teno virus (TTV), rolling-circle amplification was used to amplify DNA extracted from eight human and four pig serum samples. All samples gave high molecular weight (>30 kb) amplification products. By restriction endonuclease digestion, these products generated DNA fragments whose sizes were consistent with those of human TTV (3?8 kb) and swine TTV (Sd-TTV; 2?9 kb) genomes. Two TTV isolates derived from a single AIDS patient, as well as two Sd-TTV isolates derived from a single pig, were characterized by complete nucleotide sequencing. One of the Sd-TTV isolates showed very low (43-45 %) nucleotide sequence similarity to the other Sd-TTV isolate and to the prototype isolate Sd-TTV31, and could be considered the prototype of a novel genogroup.Torque teno virus (TTV) is a non-enveloped, singlestranded, circular DNA virus with a genomic length of 3?4-3?9 kb (Nishizawa et al., 1997;Miyata et al., 1999;Mushahwar et al., 1999) and has been recently classified into a novel, floating genus called Anellovirus (Biagini et al., 2005). TTV is found in the plasma of >80 % of the human population worldwide (Prescott & Simmonds, 1998;Takahashi et al., 1998;Niel et al., 1999). Co-infection of single individuals with multiple TTV isolates is frequent (Takayama et al., 1999;Niel et al., 2000). TTV has a wide genetic diversity and virus isolates have been classified into five main phylogenetic groups (1-5) with low nucleotide sequence similarity between them (Peng et al., 2002). Anelloviruses are not restricted to human hosts and have also been detected in non-human primates, tupaias, cats, dogs and pigs Verschoor et al., 1999;Okamoto et al., 2001Okamoto et al., , 2002. However, few complete nucleotide sequences from animal TTVs have been reported.In their natural replication cycle, some DNA viruses, like circoviruses, employ a rolling-circle mechanism to propagate their circular genomes. Multiply primed rolling-circle amplification is a novel technique able to amplify circular DNA molecules such as plasmids with great efficiency (Dean et al., 2001). The method utilizes bacteriophage phi29 DNA polymerase, a high-fidelity enzyme, with a strong strand-displacing capability, high processivity and proofreading activity (Garmendia et al., 1992;Esteban et al., 1993). Unlike PCR, the primers used in the amplification reaction are random hexamers. Previous knowledge of the nucleotide sequences to be amplified therefore is not necessary. Furthermore, phi29 DNA polymerase is very stable, with linear kinetics at 30 uC for over 12 h, eliminating the need for thermal cycling. The reaction products are high molecular weight, linear, double-stranded, tandem-repeat copies of the input DNA that can subsequently be digested with restriction endonucleases.In this study, multiply primed rolling-circle amplification was ...

Expression profile and subcellular localization of Torque teno sus virus proteins

“…TTSuVs have similar genomic organization with human-infecting TTVs but share less than 45 % nt sequence identity (Niel et al, 2005;Okamoto et al, 2002). Recent studies also demonstrated a high degree of genetic variability between TTSuV1 and TTSuV2 (Cortey et al, 2011).…”

Quantitative Detection of Porcine Torque Teno Virus in Porcine Circovirus-2–Negative and Porcine Circovirus–Associated Disease-Affected Pigs

“…Sd-TTV2p by itself composes novel TTV genogroup 2. 15 A number of researchers have reported TTV prevalence in various situations. 6,8 Coinfection with different genogroups of TTV in an individual was found to be common.…”

“…After the first finding of swine TTV in 2004, different genogroups in pigs were subsequently identified. 13,15 Porcine TTV isolates Sd-TTV31 and Sd-TTV1p have a relatively high nucleotide sequence identity (69.6%) and are classified as TTV genogroup 1. Sd-TTV31, a prototype of genogroup 1, and Sd-TTV2p, a recently sequenced swine TTV, share a low nucleotide sequence identity (44%) with Sd-TTV31.…”

Antigenic diversity and seroprevalences of Torque teno viruses in children and adults by ORF2-based immunoassays

“…The ORF2 coding sequences were amplified from plasmids containing TTV genomic DNA. The study included five strains of different species from four genogroups: Gr1 TTV3 HEL32, Gr2 TTV6 LTT6, Gr3a TTV13 3h, Gr3b TTV15 5A and Gr4 TTV29 ViPi08 (Diniz-Mendes et al, 2004;Kakkola et al, 2002;Niel et al, 2005). The details of each strain are depicted in Table S2; the primers used to amplify the ORF2 fragments are shown in Table S3.…”