[Construction of a duck hepatitis B virus YMDD mutant and identification of its resistance phenotype].

OBJECTIVE: To construct a lamivudine-resistant plasmid containing 1.2 unit genome of duck hepatitis B virus and identify its replication and drug-resistance in avian LMH hepatica cells. METHODS: The recombinant plasmid PBS-DHBV1.2 was constructed using the 1.2-genome length DHBV DNA sequence from a dimer DHBV genome with pcDNA3.1 as the template. With site-directed mutagenesis, we obtained PBS-DHBV1.2-M512V plasmids with polymerase gene mutation from PBS-DHBV1.2. Two constructed plasmids were transiently transfected into LMH cells using FuGENETM6 transfection reagent and cultured in the medium containing different concentrations of lamivudine. Southern blot hybridization was performed to detect DHBV replication intermediates. RESULTS: PCR amplification, restriction digestion and plasmid sequencing all confirmed successful construction of PBS-DHBV1.2-M512V recombinant plasmid. Southern blot analysis identified the presence of all the expected DHBV replication intermediates in LMH cells. The replication capacity of the mutant plasmid was decreased by 2.7 times compared with that of the wild plasmid. The IC(50) of lamivudine was 37.12∓8.81 ng/ml for the mutant, greater than that of the wild plasmid (10.90∓4.80 ng/ml). CONCLUSION: Compared with the wild plasmid, the mutant plasmid has a lower replication capacity and sensitivity to lamivudine in vitro.

[Stable expression of HBV C gene mutants in immortalized human B-cell lines].

OBJECTIVE: To provide an cell model of immortalized lymphoblstoid B-cell lines for studying the biological characteristics of full-length hepatitis B virus (HBV) genome carrying the hot-spot mutations V60, G87, and L97. METHODS: V60, G87, and L97 mutation points were introduced into HBV p3.8 II plasmid containing 1.2 copy of HBV genome by means of site-directed mutagenesis. The HBV genome was amplified by PCR from p3.8 II and p3.8 II-V60, G87, L97 plasmid, and the PCR product was inserted into EBO-plpp eukaryotic expression vector. The recombinant vectors and the EBO-plpp vector were transfected into immortalized human lymphoblasts with lipofectamine 2000 and selected with hygromycin. Steady expression of the target genes was determined by RT-PCR, Western blotting and microparticle enzyme immunoassay. RESULTS: DNA sequence analysis indicated that the desired mutation was introduced into wild-type HBV DNA. HBsAg, HBeAg and HBcAg could be detected in EBO-HBV-transfected cell lysate or culture supernatant. CONCLUSION: Transfectants that stably express HBV mutant antigen may provide a cell model to study the biological characteristics of HBV carrying hot-spot mutation in vitro.

[Establishment of immortalized B lymphoblast cell line from patients with chronic hepatitis B].

AIM: To establish immortalized B lymphoblast cell line (BLCL) from patients with chronic hepatitis B (CHB) in vitro. METHODS: The peripheral blood mononuclear cells (PBMC) were isolated from the patients with CHB by routine method and incubated with EB virus (EBV) in the presence of CpG DNA motifs and cyclosporin A (CysA) for about 28 days. Morphological characteristic of the established immortalized BLCL was observed by microscope and the expression of CD19 and CD23 on cellular surface was determined by flow cytometry. RESULTS: BLCL was successfully established and could be cultured and propagated for a long time in vitro. CONCLUSION: EBV-immortalized BLCL provides a resource of target cells for further research on the cellular immunity of patients with CHB.

[Cloning and sequence analysis of the genome of a strain of duck hepatitis B virus].

OBJECTIVE: To clone and analyze the genome of a duck hepatitis B virus (DHBV) strain isolated from the ducks found in the local area. METHODS: The complete genome of DHBV was amplified by PCR prior to cloning into T vector and sequencing, with also homology and phylogenetic analyses. RESULT: Genome comparison of an isolated DHBV strain and 16 DHBV strains in GenBank revealed a homology of 89.4% to 99.3% at the nucleotide level, and the amino acid identity of the 3 open reading frames showed that the P region harbored more obvious variations than the other regions. CONCLUSION: The isolated DHBV strain might belong to a subtype of the virus found in the western countries.

[Cloning and sequence analysis of a novel TT virus varian].

OBJECTIVE: To clone the DNA of a TT virus (TTV) variant isolated from a patient with elevated alanine transaminase (ALT) of unknown etiology, and conduct sequence analysis. METHODS: The long fragment of TTV DNA was amplified by nested PCR and then cloned into pGEM-T vector. A clone named 56-B containing 3.2 kb TTV DNA was selected for sequence analysis besides homology analysis with other 5 TTV variants retrieved from GenBank, and phylogenetic analysis was carried out by maximum likelihood method. RESULTS: The nucleotide identities of 56-B with the other 5 TTV strains TA278, JA10, US35, SANBAN and TUS01 were 42.4%, 48.2%, 47.9%, 49.8 % and 61.7 % respectively, and the corresponding amino acid identities were even lower. Phylogenetic analysis showed that 56-B was far from other TTV strains in genetic distance that ranged from 0.344 to 0.458. However, the sequences in the 5'- and 3'-end were still much conservative. CONCLUSION: The isolated 56-B showed high heterogeneity in genetic background and was therefore quite distinct from other TTV strains as a novel TTV variant that represents a new TTV genotype.