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 ...

Hepatitis B virus (HBV) genotype A has been divided recently into two subgroups, designated A-A' (genotype A excluding A') and A'. Isolates belonging to subgroup A' have been identified in Africa. A new genotyping method, based on PCR amplification of the pre-S/S genome region and subsequent restriction fragment length polymorphism (RFLP) analysis, was developed, that established a correlation between RFLP subtypes and subgroups within genotype A. To investigate the occurrence of subgroup A' in South America, 119 Brazilian HBV isolates were analyzed. Ninety-three (78%) of them belonged to genotype A, with three predominating RFLP subtypes: 44 (37%) isolates were classified as AI, 30 (25%) were AII, and 18 (15%) were AIII. Pre-S/S nucleotide sequences of 15 genotype A isolates were determined. Phylogenetic analysis performed with these 15 and an additional 41 sequences revealed that isolates AI and AII clustered in subgroup A', whereas isolates AIII were classified into subgroup A-A'. The correlation RFLP subtypes-subgroups was confirmed by the presence of amino acid residues specific for subgroup A' in the surface antigens and polymerase of isolates AI and AII. The high proportion (63%) of isolates from subgroup A' suggested an African origin for a large number of Brazilian HBVs.

Background: Lamivudine is an oral nucleoside analogue widely used for the treatment of chronic hepatitis B. The main limitation of lamivudine use is the selection of resistant mutations that increases with time of utilization. Hepatitis B virus (HBV) isolates have been classified into eight genotypes (A to H) with distinct geographical distributions. HBV genotypes may also influence pathogenic properties and therapeutic features. Here, we analyzed the HBV genotype distribution and the nature and frequency of lamivudine resistant mutations among 36 patients submitted to lamivudine treatment for 12 to 84 months.

Hepatitis A virus (HAV) isolates from around the world have been classified into seven genotypes (I-VII). Most human strains belong to genotype I, which has been divided into two subgenotypes, A and B. South America has provided a small number of strains studied at the genome level. In the present study, IgM anti-HAV antibodies were detected in 116 out of 250 (46%) serum samples collected from consecutive patients with acute hepatitis referred to the Brazilian Reference Center for Viral Hepatitis, Rio de Janeiro. Viral RNA were extracted from all 250 samples and submitted to a reverse transcription-polymerase chain reaction (RT-PCR) assay designed to amplify a genome segment in the VP1/2A junction region. HAV RNA was detected in 54/116 (47%) and 17/134 (13%) IgM anti-HAV-positive and -negative sera, respectively. In addition, HAV RNA was detected in 17/35 (49%) IgM anti-HAV-positive sera that had been collected at a day care center where cases of acute hepatitis were being observed for 3 months. Nucleotide sequences (168 bp) of PCR products were determined for 30 HAV isolates. Phylogenetic analysis showed that 21 belonged to subgenotype IB, while 9 were of subgenotype IA. Interestingly, a concomitant circulation of isolates from subgenotypes IA and IB was observed in the day care center.

This study was conducted in an Afro-Brazilian, slave-descendant community with high (42.4%) hepatitis B virus (HBV) prevalence. Twenty (8.4%) out of the 239 subjects under study were HBsAg-positive, and HBV-DNA was detected in 59 (25%) individuals. A high rate (18.3%) of occult infection was therefore observed that was associated to low HBV loads (mean, 1.8 x 10(4) copies/ml) and to a specific amino acid substitution (C100Y) in the small surface antigen. Genotyping of 50 isolates showed that 43 (86%) were of subgenotype A1, one (2%) from subgenotype A2, and five (10%) from subgenotype D. Mixed genotypes A1 and E were observed in one (2%) sample. The genetic distance (0.8 +/- 0.3%) among the HBV/A1 isolates from the community was smaller than the intragroup divergence among A1 isolates from Brazil as a whole, but it was similar to that found between A2 isolates from different countries, suggesting that HBV/A1 was introduced in the community through different sources. The substitution W501R (polymerase), previously reported only in Gambia, was observed in 46% of the HBV/A1 isolates. The precore/core promoter region of HBsAg-positive isolates showed several substitutions that could explain the anti-HBe phenotype found in 18 of 20 (90%) of the HBsAg-positive subjects.

Brazil is a country of low hepatitis B virus (HBV) endemicity in which the genotype A of HBV (HBV/A) is the most prevalent. The complete nucleotide sequences of 26 HBV/A isolates, originating from eight Brazilian states, were determined. All were adw2. Twenty-three belonged to subgenotype A1 and three to A2. By phylogenetic analysis, it was shown that all the 23 HBV/A1 isolates clustered together with isolates from Bangladesh, India, Japan, Nepal, the Philippines and United Arab Emirates, but not with those of Congo, Kenya, Malawi, Rwanda, South Africa, Tanzania, Uganda and Zimbabwe. Four amino acid residues in the polymerase (His138 in the terminal protein domain, Pro18 and His90 in the spacer, and Ser109 in the reverse transcriptase), and one (Phe17) in the precore region, predominated in Latin American and Asian HBV/A1 isolates, but were rarely encountered in African isolates, with the exception of those from Somalia. Specific variations of two adjacent amino acids in the C-terminal domain of the HBx protein, namely Ala146 and Pro147, were found in all the Brazilian, but rarely in the other HBV/A1 isolates. By Bayesian analysis, the existence of an ‘Asian-American’ clade within subgenotype A1 was supported by a posterior probability value of 0.996. The close relatedness of the Brazilian, Asian and Somalian isolates suggests that the HBV/A1 strains predominant in Brazil did not originate from the five million slaves who were imported from Central and Western Africa from 1551 to 1840, but rather from the 300–400,000 captives forcibly removed from southeast Africa at the middle of the 19th century.

TT virus (TTV) is an unenveloped, single-stranded DNA virus that was discovered recently in the sera of Japanese patients with posttransfusion hepatitis of unknown etiology. A high prevalence of TTV infection in blood donors of several countries, including Brazil, has been demonstrated. To study the variation in TTV prevalence between different age groups, sera from 223 individuals without liver disease, aged 0-80 years, were tested by the polymerase chain reaction for the presence of TTV DNA. All subjects were inhabitants of the city of Rio de Janeiro, Brazil. The prevalence increased continuously with age (P <.001), from 17% among children under the age of 11 years, to 57% in people older than 50 years. To assess vertical transmission, sera from 105 unselected, consecutive parturient women attending a public maternity hospital were paired with cord bloods and examined for the presence of TTV DNA. Thirty-seven (35%) mothers were found to be TTV infected. Seven cord bloods were also positive, suggesting the possible transplacental transmission of the virus. Furthermore, a direct correlation between TTV viremia and presence of antibodies to the enterically transmissible hepatitis A virus (HAV) was observed in this group of women, with a relative risk of TTV infection of 5.09 (95% confidence interval 0.76-34.03) for women with anti-HAV, compared with women without. This finding suggested that the fecal-oral route might be an important route of TTV transmission.

Hepatitis B virus (HBV) genomes have been classified into six groups, A-F, group F being the most divergent. South America has provided the smallest number of strains studied at the genome level. The only HBV strain from this region sequenced completely has been classified in group F, and contains the most divergent of the HBV genomes presently known [Naumann et al. (1983): Journal of General Virology 74:1627-1632]. To evaluate genetic relatedness between strains isolated in South America and in the rest of the world, a restriction endonuclease analysis was carried out on 14 HBV strains (4 adw2, 3 adw4, 3 ayw2, and 4 ayw3) isolated in Rio de Janeiro, Brazil. C, pre-S, and X genes along with the 5' part of the P gene from these strains were amplified by the polymerase chain reaction. The DNA fragments were digested by BamHI, BstEII, EcoRI, HhaI, and TaqI endonucleases. The restriction patterns obtained were compared with those deduced from the nucleotide sequence of 26 HBV strains isolated in other continents. The results showed a large genetic variability of Brazilian strains. Taking into account that all the samples examined possessed the w antigenic subdeterminant, the number of different restriction patterns of strains isolated in Rio de Janeiro was at least as large as that of the reference strains isolated in the rest of the world. Some original restriction patterns were found in adw4 and ayw2 HBV strains.