RAPID DETECTION OF HUMAN TORQUE TENO VIRUSES USING HIGH-RESOLUTION MELTING ANALYSIS
Spandole S1*, Cimponeriu D1, Toma M1, Radu I1, Ion DA2
*Corresponding Author: Ms. Sonia Spandole (Ph.D. Student), Department of Genetics, University of Bucharest, Intrarea Portocalelor Street, No 1-3, 060101, Bucharest, Romania; Tel.: 004-0764-824-281, Fax: 004-0213-181- 565; E-mail: sonia.spandole@gmail.com
page: 55

INTRODUCTION

Torque teno viruses (TTVs) are small, non enveloped viruses, with an icosahedral capsid and a singlestranded circular DNA genome. One characteristic of TTVs is the extreme genomic heterogeneity that is highly uncommon for DNA viruses. Three virus species that infect humans have now been identified: TTV (Torque teno virus) [1], TTMV (Torque teno mini virus) [2] and TTMDV (Torque teno midi virus) [3]. Torque teno mini virus (2.8-2.9 kb) and TTMDV (3.2 kb) have smaller genomes than the TTV genome (3.75-3.9 kb). The TTV, TTMV and TTMDV each consist of a wide repertoire of isolates with genomewide sequence divergences of at least 50.0, 40.0 and 33.0%, respectively [4]. Based on genomic heterogeneity, TTVs were separated into 39 genotypes and five major genetic groups [5,6], and it was suggested that these viruses may exist as a swarm of closely related but different viral species [7]. The sequence divergence is not evenly distributed throughout the genome. The untranslated region (UTR) is well conserved (73.0% nucleotide identity between the two highly divergent isolates TA278 and SANBAN vs. 57.0% for the entire genome) and contains several highly con-served sequences (with more than 90.0% identity between isolates). In contrast, the translated region is characterized by a very high degree of diversity [8,9]. The TTVs disease-causing potential is still under debate. Rocchi et al. [10] showed that certain variants pre-sent CpG motifs that are activators of proinflammatory cytokine production via toll-like receptor 9, and thus, may increase severity of inflammatory diseases. For the identi-fication of these variants, new methods for assessing the viral diversity are needed. Polymerase chain reaction (PCR)-based methods were used to detect the presence of TTVs DNA in biological samples. The high TTVs sequence divergence and the particularities of PCR assay used for detection (primers, genomic region amplified, protocols) [11] may determine preferential amplification of some TTV genotypes and, thus, can influence the prevalence rates of TTVs presented in different articles. One of the first diagnostic assays for TTVs was based on PCR that used primers derived from the N22 region of ORF1 [1]. This primer set did not amplify all TTV phylo-genetic groups. Subsequently, PCR techniques aimed for the conserved UTR, near the 5 end of ORF2 (Takahashi et al. [12]) or near the 3 end of ORF3 (Leary et al. [13]). Methods based on analysis of changes in fluorescence intensity contribute to genome diversity analysis. One of these methods is high-resolution melting analysis (HRMA) [14], which uses high datadensity acquisition during melting of amplicons in the presence of intercalating dye. Because of its low-cost, speed and simplicity, this method is used for genetic discrimination of various microorganisms, including viruses [15-18] and bacteria [19,20]. However, as far as we know, it has never been used for TTVs genotyping. The aim of this study was to assess if HRMA can be used for efficient and rapid detection of TTVs variants.



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