These samples were referred to the public central Noel Nutels laboratory in Rio de Janeiro, Brazil, for the assessment of HBV loads. Individuals with clinical symptoms of acute hepatitis were monitored in the Viral Hepatitis Ambulatory Center of our Institution. The diagnosis of acute HBV infection was confirmed by positivity to anti-HBc IgM antibodies (AxSYM CORE-M; Abbott, Delkenheim, Germany). Twenty samples

from these individuals were included in the present study. The research use of these samples was approved by the Fiocruz Ethics Committee, and written informed consent was obtained from all subjects. HBV direct sequencing and HBV quantification by real-time PCR HBV DNA was extracted

from serum samples using the High Pure Viral Nucleic Acid S3I-201 KPT-8602 order kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Viral DNA was eluted in 50 μL of Elution Buffer. For the direct Sanger sequencing method, the pre-S/S genome region was amplified by semi-nested PCR. The first-round PCR product was amplified with the primer pair PS1 and P3, and the second round was performed using the sense primer PS1 and a mixture of two antisense primers, S2 and S22, as previously described [22]. DNA was amplified using 5 U/μL Taq DNA polymerase (Invitrogen, San Diego, CA, USA) and 10 mM dNTPs in a final volume of 50 μL. First round PCR was performed using the following conditions: 94°C for 3 min (initial denaturation), then 30 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min 30 s, followed by a final

elongation step (7 min at 72°C). Second-round thermocycling conditions were 94°C for 3 min, then 30 cycles of 95°C for 30 s, 52°C for 10 s and 72°C for 2 min, followed by a final elongation step (7 min at 72°C). The lower limit of detection of the PCR assay was 100 copies/mL. PCR products were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA), and were prepared for sequencing using a Big Dye Terminator 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with external primers PS1 and S2 or S22, internal sense primer S4 (5′-TGCTGCTATGCCTCATCTTCT-3′; nucleotides check [nt] 416-436) and antisense primer S7 (5′-TGAGCCAGGAGAAACGGGCT-3′; nt 676-656). The sequence was PXD101 in vivo determined by separation and analysis of extension products using an automated ABI 3730 DNA Analyzer (Applied Biosystems). HBV genotyping was performed by phylogenetic analysis of the pre-S/S gene of the sequences determined in this study in the context of HBV sequences representing all known genotypes available in GenBank. Sequences were aligned using the ClustalW program [23], and the phylogenetic tree was generated using the neighbor-joining method (bootstrap resampling test with 1,000 replicates) in MEGA version 4.0 software [24].