OBJECTIVETo clone and analyze duck hepatitis B virus genome from Chongqing brown duck.

METHODSDuck hepatitis B virus (DHBV) DNA extracted from a Chongqing brown duck was amplified by PCR and cloned into PGEM-T vector using T-A clone method. The sequence of this DHBV genome was analyzed with some softwares after identified.

RESULTSThe duck hepatitis B virus genome from Chongqing brown duck (DHBVcq), which was 3 024 nucleotides long, contained three ORFs whose onset and end nucleotides were in accord with those of HPUGA, encoding P, PreC/C and PreS/S protein respectively. Comparison of this strain with other DHBV reported in GenBank showed that the homology of DHBVcq and M32990 got the highest score of 94.9% at nucleotide level, while DHBVcq and DHBVCG got the least (89.8%). Most of the conserved regulation nucleotides and amino acids sequence found in other DHBV were also identified in DHBVcq. The epsilon region of DHBVcq, which was important for encapsidation of pgRNA and synthesis of minus-strand DNA, differed from that of most other DHBV strains, forming a stem-loop conformation with a three- nucleotides upper stem rather than a common nine-nucleotides one in free status.

CONCLUSIONThe successful clone and analysis of DHBVcq provide further studies with helpful information.

Animals ; Cloning, Molecular ; DNA, Viral ; chemistry ; genetics ; Ducks ; Hepatitis B Virus, Duck ; classification ; genetics ; Hepatitis Virus, Duck ; genetics ; Open Reading Frames ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sequence Homology

To develop a reverse dot blot assay for rapid detection of HBV genotypes.Specific oligonucleotides probes were desighed and immobilized on nylon membranes.The DNA sample to be tested was PCR-amplified with DIG labeling primers and then hybridized with the immobilized probes.This procedure for detecting HBV genotypes was simple,rapid and specificity.30 specimens in Chongqing area were collected and detected by this method,and results were evaluated using direct sequencing.Results showed that: This new method was applicable to precise detection HBV genotypes for specimen with copies up to 103,and the HBV genotyping results showed that genotype B was the predominant genotype in Chongqing area.

In order to demonstrate PTB bind to HPRE,reverse transcription,PCR-mediated detection,were used.HepG2.2.15 cell line and HBs-HPRE transient expression cells were adopted to identify PTB function in HBV life cycle.The results showed that PTB could directly bind to HPRE RNA.Functional analysis indicated that PTB could inhibit the expression of HBs antigen and this inhibition was in a dose-dependent manner in HepG2.2.15 cells.Higher expression of HBs in cells transfected pcDNA3-HBs-HPRE comparing with pcDNA3-HBs,and this high expression could also be inhibited by PTB.The data demonstrated that PTB inhibits HBs expression by interacting with HPRE.

Objective To study the effect of early care intervention on the myocarditis patients suffering from acute upper respiratory tract infection.Methods Totals of 78 patients were randomly divided into two groups,each with 39 cases.The control group received conventional care,while the intervention group received early nursing intervention on the basis of routine care.Results After two weeks,the total effective rate of the intervention group was 97.4％,higher than 87.2％ in the control group,and the difference was statistically significant (Z =2.434,P ＜ 0.05).The cTnI and myocardial enzymes of the two groups were significantly decreased compared with before (P ＜ 0.05).And indicators of intervention group decreased significantly compared with that of the control group (t =4.189,3.289,3.260,3.488,respectively ; P ＜ 0.05).On the care evaluation,the total satisfaction of the control group was 84.6％ (33/39),which was lower the intervention group that was 97.4％ (Z =3.017,P ＜ 0.05).Conclusions Early care intervention on the myocarditis patients suffering from acute upper respiratory tract infection has positive clinical value.

OBJECTIVETo investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication.

METHODSHepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference.

RESULTSouthern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity.

CONCLUSIONHS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.

DNA Replication ; DNA, Viral ; biosynthesis ; Hep G2 Cells ; Hepatitis B virus ; genetics ; physiology ; Humans ; Plasmids ; Sulfotransferases ; genetics ; Transfection ; Virus Replication

OBJECTIVETo establish a stable cell line that can replicate hepatitis B virus (HBV) DNA carrying the reverse transcriptase sequence derived from a clinical isolate.

METHODSNested PCR was used to amplify the HBV DNA fragment from the serum. The fragment was cloned into a plasmid that can support HBV replication in vitro by fragment substitution reaction (FSR), followed by the cloning of the neomycin expressing fragment downstream from HBV DNA. G418 selection was conducted after the transfection of HepG2 cells with the recombinant DNA. Real-time PCR and enzyme linked immunosorbent assay (ELISA) were used to screen stable cell lines that can replicate HBV DNA, and the replication of HBV DNA by the cell line was confirmed by using Southern blot analysis.

RESULTSFragment nt55-1654 amplified from the serum DNA was substituted to the plasmid pLL, generating the plasmid p11. The neomycin fragment was cloned into p11, leading to the plasmid p11-neo, and p11-neo was confirmed to be HBV-replication-competent. A stable cell line named 3-10 that can replicate HBV DNA was obtained.

CONCLUSIONSA stable cell line was established that can replicate HBV DNA carrying the reverse transcriptase sequence derived from a clinical isolate. Real-time PCR plus ELISA may help to rapidly screen out stable cell lines replicating HBV DNA.

Cell Line ; Cloning, Molecular ; DNA Replication ; DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Hepatocytes ; cytology ; virology ; Humans ; Plasmids ; RNA-Directed DNA Polymerase ; genetics ; Virus Replication ; genetics

HBV infections leads to severe public health problems around the world, especially in China. Improved understanding of the molecular mechanisms of HBV reverse transcription is fundamental for optimization of treatment and solution to drug-resistance. Recently, the main structural basis involved in the process of HBV reverse transcription and the cis-elements were revealed by means of biochemistry and genetics. The entire process of reverse transcription is completed mainly through the first template switch mediated by the P- epsilon structure; the second template switch mediated by 5E/3E and M structure; and the third template switch mediated by 5' r / 3' r structure. The important structure and the cis-elements involved in this process are the focus of this review, at the same time, an overview of the progress in relevent studies is demonstrated to show the whole picture of the HBV reverse process.
Animals ; Hepatitis B ; virology ; Hepatitis B virus ; enzymology ; genetics ; metabolism ; Humans ; RNA, Viral ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Viral Proteins ; genetics ; metabolism

OBJECTIVESTo establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues.

METHODSAccording to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively.

RESULTSThe specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing.

CONCLUSIONReverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.

Amino Acid Substitution ; Antiviral Agents ; pharmacology ; DNA, Viral ; genetics ; Drug Resistance, Viral ; drug effects ; genetics ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Mutation ; Nucleic Acid Hybridization ; methods

OBJECTIVETo establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system.

METHOD26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively.

RESULTSensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test.

CONCLUSIONThough the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

DNA, Viral ; genetics ; Drug Resistance, Viral ; genetics ; Hepatitis B virus ; drug effects ; genetics ; Humans ; Hybridization, Genetic ; Mutation ; Nucleic Acid Hybridization ; methods ; Oligonucleotide Array Sequence Analysis ; Sensitivity and Specificity

OBJECTIVETo investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.

METHODSThe PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.

RESULTSThe transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.

CONCLUSIONSoluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Escherichia coli ; metabolism ; Genetic Vectors ; Hepacivirus ; Recombinant Proteins ; genetics ; metabolism ; Viral Core Proteins ; biosynthesis ; genetics ; metabolism ; Viral Nonstructural Proteins ; metabolism