HBV genotyping was performed by sequencing and phylogenetic analyses of the surface (S) and core (C) fragments. Briefly, HBV DNA was first extracted from 400 \u00b5L of plasma and eluted in 100 \u00b5L\u00a0 of pure water, using the QIAamp Viral DNA Mini Kit (QIAGEN, Courtaboeuf, France) followed by semi-nested PCR amplification of the S (930 bp) and C (1010 bp) gene fragments using MP Taq Core Kits 25 (MP Biomedical Diagnostic, Europe). The S fragment amplification was performed as described elsewhere.(Hu X, et al., 2000 [PMID:\u00a010677515] ; Makuwa M et al., 2006 [PMID: 16847965]; Olinger CM et al.,\u00a02006 [PMID: 16603517])The first round was performed using primers sets 58P (5\u2019-CCT GCT GGT GGC TCC AGT TC-3\u2019) and 979 (5\u2019-ATT GGA AAG TAT GTC AAA GAA TTG TGG GTC TTT TG-3\u2019). The 50 \u00b5L final reaction mixture contained 31.4 \u00b5L of RNase DNase Free water, 5 \u00b5L of buffer 10X with MgCl2 (25 mM), 0.4 \u00b5L of dNTPs (25 mM), 1.5 \u00b5L of each primer (10 \u00b5M), 0.2 \u00b5L of Taq polymerase (5 U\/\u00b5L) and 10 \u00b5L of extracted DNA. The second round PCR used the 58P\/Mc2r (5\u2019-TGGAAGTTGGGGATCATTGCC-3\u2019) primer on a 50 \u00b5L final reaction mixture containing 36.4 \u00b5L of RNase DNase Free water, 5 \u00b5L of buffer 10X with MgCl2 (25 mM), 0.4 \u00b5L of dNTPs (25 mM), 1.5 \u00b5L of each primer (10 \u00b5M), 0.2 \u00b5L of Taq polymerase (5 U\/\u00b5L), and 5 \u00b5L of first round PCR product. The PCR program was the same for the first and second round PCRs\u00a0 including denaturation at 94\u00b0C for 5 min followed by 40 cycles of denaturation at 94\u00b0C for 1 min, annealing at 55\u00b0C for 30 s and elongation at 72\u00b0C for 1 min, followed by final elongation at 72\u00b0C for 5 min.\u00a0 For the C fragment amplification, the couplet of primers BCP1F (5\u2019-GCA TGG AGA CCA CCG TGA AC-3\u2019) \/ 2853N (5\u2019-TCA CCA TAT TCT TGG GAA CA-3\u2019) was used for the first round and the couplet BCP2F\u00a0(5\u2019-CAT AAG AGG ACT CTT GGA CT-3\u2019) \/ 2853N for the second round. The amplification conditions were the same as for the S fragment.\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0The PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France) and submitted for sequencing at Macrogen Inc (Meibergdreef, Netherlands).