Torque teno virus is a human non-enveloped single- stranded circular DNA virus with icosahedral symmetry

Torque teno virus is a human non-enveloped singlestranded circular DNA virus with icosahedral symmetry and a particle size of approximately 30 nm. TTV was first characterised as a blood-borne virus and was initially referred to as transfusion-transmitted virus (Nishizawa et al. 1997). Its negative-sense genome is 3.6-3.9 kb in size (Mushahwar et al. 1999). Recently, it was renamed Torque teno virus (TTV) and classified as a member of the genus Alphatorquevirus in the family Anelloviridae (ICTV Virus Taxonomy 2009) (ictvonline.org/). Although TTV infection has been suggested to be associated with a number of diseases based on epidemiological data, there is no direct causal evidence linking TTV infection to specific clinical manifestations. This virus is distributed around the world and the prevalence of TTV infection is similarly high in all groups tested, including patients with liver (Asim et al. 2010), acute respiratory (Maggi et al. 2003) and renal diseases (Irshad et al. 2010), as well as in human immunodeficiency virus (HIV)-positive subjects (Devalle & Niel 2004), drug users (Alzahrani et al. 2009) and healthy individuals (Vasilyev et al. 2009).The objective of the present study was to determine the prevalence of TTV DNA in blood donors and patients requiring haemodialysis in the southern state of Rio Grande do Sul (RS), Brazil. A cross-sectional study was conducted from May-August 2010. Plasma samples from 150 consecutive blood donor candidates and all 77 patients receiving regular haemodialysis treatment at one dialysis unit in the city of Pelotas (in the southern region of RS) were tested for the presence of TTV DNA. This study was approved by the Research Committee of the Lutheran University of Brazil (2009440H). All participants signed an informed consent form and filled out an epidemiological questionnaire. Hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (HCV) and anti-HIV I/II antibodies were detected using standard commercially available assays. The alanine aminotransferase (ALT) serum levels were measured in the patients requiring haemodialysis. Blood was collected and viral DNA was extracted according to the method of Niel et al. (1994). TTV DNA was amplified in a sensitive, single-step polymerase chain reaction (PCR) assay. Oligonucleotide primers were designed to hybridise to the untranslated region, which is the most conserved region of the TTV genome. PCR amplification was then conducted with the TTV1 (sense, 5’-TGCACTTCCGAATGGCTGAGTT-3’, designed for this study; nt 94-115) and TTV NG0147 (antisense, 5’-GCCAGTCCCGAGCCCGAATTGCC-3’; nt 209-231) primers (Okamoto et al. 1999). The resulting amplicon was 136 bp in length. The indicated base positions of the primers were based on the nucleotide sequences described by Niel et al. (2005), which are available in GenBank (accession AY823989). The sensitivity of the primer pair was determined by testing the amplification of 10-fold serial dilutions of plasmid 3h (Niel et al. 2005), which carries the entire TTV genome, yielding a detection limit of 10 femtograms. These experiments were repeated three times. This plasmid was also used as a positive control in the PCR assays. The selected primer sequences were examined for similarities to sequences from other organisms using NCBI BLAST and a high level of specificity was found. Water was used as a negative control. The amplification was performed using 3 μL of DNA and 2.5 units of Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) in a final volume of 50 μL. AfFinancial support: ULBRA/RS + Corresponding author: [email protected] Received 2 October 2011 Accepted 11 January 2012 The high prevalence of Torque teno virus DNA in blood donors and haemodialysis patients in southern Brazil