Serum and plasma samples
Blood samples were drawn from 21 asymptomatic blood donors, at the Central Blood Bank, Centro Médico Nacional-Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS), and from 12 symptomatic patients attending the Hepatitis Clinic, Gastroenterology Service, General Hospital, CMN-SXXI-IMSS. All 33 patients were selected because they were known to be HBsAg carriers. Symptomatic cases were receiving lamivudine (LMV) treatment during at least one year. The study was approved by the IRB Committee of IMSS. Informed consent was obtained from all studied participants.
Serology
Assay for HBsAg was performed in sera using a commercial HBsAg test (version 2 IMx, Abbott Laboratories, IL, USA) following manufacturer's instructions. Hepatic function tests (ALT, AST, Billirubins) were performed using the commercially available Dimension® Clinical Chemistry System (Dade Behring, Inc. Neward, DE, USA).
Viral load
HBV viral load was determined in plasma with a commercial quantitative assay (COBAS AMPLICOR HBV Monitor™, Roche Diagnostics, Indianapolis, IN, USA) according to manufacturer's instructions.
DNA extraction
HBV DNA was extracted from plasma specimens with QIAamp® Ultrasens® Virus kit (QIAGEN) following manufacturer's instructions.
PCR amplification of the S gene
A fragment of the S gene was amplified by nested PCR. Sequences of the primers and their relative positions were as follows: outer primers included HBV1-sense, 5'-CGC TGG ATG TGT CTG CGGCGT-3', position 371–391, and HBV2-antisense, 5'-CGA ACC ACT GAA CAA ATG GCA CT-3', position 682–704; inner primers were HBV3-sense, 5'-CAT CCT GCT GCT ATG CCT CAT CT-3', position 409–431 and HBV4-antisense, 5'-GGC ACT AGT AAA CTG AGC CA-3', position 668–687 (30). Ten μL of DNA extracted from plasma (template) were mixed with 40 μL of reaction mixture containing 1× PCR Buffer, 50 pmoles of each primer, 0.8 mM dNTPs, 2.5 mM MgCl2, and 2.5 U Taq polymerase (Amplificasa® BIOGÉNICA, Mexico City, Mexico). Annealing and elongation for both primer pairs was for 90 sec at 47°C and 72°C, respectively. The first PCR was carried out for 45 cycles and the nested PCR for 40 cycles, using an initial denaturing step of 95°C for 2 min and a final amplification step of 72°C for 15 min [39].
Sequencing
PCR products (279 bp) were run in 2% agarose gel electrophoresis and the isolated band was extracted with a commercial kit (QIAquick® Gel Extraction Kit, QIAGEN). The purified product was used for sequencing with the chain termination method, using Big Dye Terminator version 3.1 (ABI PRISM™, Foster City, CA, USA). Extension products were purified (DyeEx™ 2.0 Kit, QIAGEN) and separated in an automated DNA genetic analyzer (377 ABI PRISM™).
Sequence analysis
Alignment of sequences was done using the Mega 3.1 program and analyzed manually by visual inspection.
Genotyping
Genotyping was carried out in DNA extracted from plasma samples, using the INNO-LiPA HBV amplification and genotyping tests (Innogenetics, Ghent Belgium), following manufacturer's instructions.
NCBI genotyping system
Genotype was also determined by sequence analysis of the 279 bp fragment from HBV "a" determinant, using the genotyping tool available at the National Library of Medicine's National Center for Biotechnology Information (NCBI) [40] using BLAST with a set of reference sequences with known genotypes [41].
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