Glucose Homeostasis Is Dysregulated in Ducks Infected with Duck Hepatitis B Virus.

INTRODUCTION: The association between hepatitis B virus (HBV) infection and the development of diabetes remains controversial. This study examined the effect of HBV infection on glucose homeostasis using a duck HBV (DHBV) model. METHODS: Plasma DHBV DNA was detected by quantitative polymerase chain reaction (PCR). Tissue infection of DHBV was determined by detecting DHBV covalently closed circular DNA (cccDNA) with a method of rolling circle amplification combined with cross-gap PCR, and verified by fluorescence in situ hybridization assay. An intravenous injection glucose tolerance test (GTT) was used to analyze the effect of DHBV infection on glucose tolerance. RESULTS: Of the finally included 97 domestic ducks, 53 (54.6%) were congenitally infected by DHBV. The positive rate of DHBV cccDNA in the liver, kidney, pancreas, and skeletal muscle of the infected ducks was 100, 75.5, 67.9, and 47.2%, respectively. The DHBV-infected ducks had higher blood glucose levels at 15 and 30 min post-load glucose (p < 0.01 and p < 0.001, respectively) in the GTT, much more individuals with greater glucose area under curve (p < 0.01), and a 57% impaired glucose tolerance (IGT) rate, as compared with noninfected controls. In addition, the subgroups of the infected ducks with DHBV cccDNA positive in skeletal muscle maintained the higher blood glucose level up to 2 h post-load glucose during the GTT and had a 76% IGT rate. CONCLUSION: These results suggest that DHBV intrahepatic and extrahepatic infection impairs glucose tolerance, and thus evidence the association of DHBV infection with the dysregulation of glucose metabolism.

Anti-hepatitis B virus activity and hepatoprotective effect of des(rhamnosyl) verbascoside from Lindernia ruellioides in vitro.

Although clinically approved hepatitis B virus (HBV) polymerase inhibitors (lamivudine-3TC, entecavir, etc.) serve as effective therapeutics, the virus can easily generate resistance to them. Therefore, the treatment of HBV infection remains a public health problem. Numerous studies have shown that natural products have prospective anti-HBV activity. The purpose of this study was to isolate and extract des(rhamnosyl) verbascoside from Lindernia ruellioides (Colsm.) Pennell and explore its anti-HBV and hepatoprotective effects. Anti-HBV activity was evaluated in HepG2.2.15 cells, a human hepatocellular carcinoma cell line with HBV-stable infection, and its protective effect was evaluated in HL-7702 cells, a normal human liver cell line. HepG2.2.15 cells maintained normal growth morphology within the selected concentration range of des(rhamnosyl) verbascoside. It also inhibited the expression of HBV antigens and HBV DNA in a dose- and time-dependent manner in vitro. Further, western blot experiments showed that it could downregulate HBV X protein (HBx) expression in a dose-dependent manner. In the H2 O2 -induced hepatocyte injury model, the cell-survival rate of the HL-7702 cells with the highest drug dose reached 85.25%, which was significantly improved compared with that of the model group. Most of the cells returned to normal morphology, showing polygonal or fusiform structures. Thus, it may be stated that des(rhamnosyl) verbascoside exhibits anti-HBV activity and hepatoprotective effects in vitro and may exert an anti-HBV effect via antigen inhibition, HBV DNA secretion, and HBx protein expression.

Hepatitis B virus rtA181T/sW172non-stop mutation may increase resistance fold to adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation.

The study aimed to characterize rtA181T/sW172stop (*) and rtA181T/sW172non-stop mutations of hepatitis B virus (HBV). Total of 22,009 patients who visited Beijing 302 Hospital from 2007 to 2016 were enrolled. These patients all received nucleos(t)ide analogues (NAs) treatment and their serum samples were collected for sequence analysis of HBV reverse-transcriptase (RT) and S regions. The rtA181T mutation was detected in 5.37% (1182/22,009) of the patients' samples. The rtA181T-causative sW172*, sW172non-stop (sW172 L/S), and mixed sW172*/non-stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively. The patients with rtA181T/sW172non-stop mutants had a higher HBV DNA level compared to those with rtA181T/sW172* mutants. 44.33% (524/1182) rtA181T-positive samples were detected with signature drug-resistant mutations, including 325 with adefovir-resistant mutation rtA181V/N236T, 57 with lamivudine-resistant mutation rtM204V/I, 99 with entecavir-resistant mutation rtM204V/I plus rt184/202/250 substitution(s), and 43 with multidrug-resistant mutation rtA181V/N236T + rtM204V/I ± rt184/202/250 substitution(s). The rtA181T/sW172non-stop mutation had a higher ratio of coexistence with adefovir-resistant mutation compared to rtA181T/sW172* mutation (42.86% vs. 24.59%, P < 0.05). rtA181T/sW172S + rtN236T and rtA181T/sW172L + rtN236T mutants exhibited higher HBV DNA production and adefovir resistance fold than that of rtA181T/sW172* + rtN236T mutant (98.02% and 85.5% vs. 42.1% in HBV DNA production, and 7.38-fold and 5.49-fold vs. 3.69-fold in half maximal effective concentration of wild-type strain); rtA181T/sW172L + rtS202G + rtM204V strain exhibited higher HBV DNA production and entecavir resistance fold than that of rtA181T/sW172* + rtS202G + rtM204V strain (50.98% vs. 34.49%, 524.00-fold vs. 69.33-fold). In conclusion, rtA181T/sW172non-stop mutation may increase resistance fold of adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation and might influence clinical presentation of NAs-treated patients.

Identification of a novel human estrogen receptor-α splice variant able to enhance malignant biological behaviors of breast cancer cells.

Since the early 1990s, multiple human estrogen receptor-α (hER-α) splice variants have been identified, of which the majority contain ≥1 deleted exon, and some are expressed as proteins with modified functions from the wild-type 66 kDa hER-α (ER-α66). In the present study, a novel hER-α splice variant, ER-α30, was identified and cloned from clinical breast cancer tissue. The ER-α30 sequence lacked a ligand-binding domain and a ligand-dependent transcriptional activation domain but retained the N-terminal transcriptional activation domain, the DNA-binding domain and a partial hinge domain, and possesses a unique 10-amino-acid domain. The expression of ER-α30 was associated with ER-α66-negative and progesterone receptor-negative breast cancer. The 30 kDa protein was expressed in stably transfected MDA-MB-231 cells, and the overexpression of ER-α30 in MDA-MB-231 cells enhanced malignant biological behaviors, including cellular proliferation, migration and invasion in vitro. The results of the present study indicated that ER-α30 might represent a potential biomarker for breast cancer.

Molecular and biochemical characterization of squalene synthase from Siraitia grosvenorii.

OBJECTIVES: To clone and characterize the squalene synthase from Siraitia grosvenorii (SgSQS). RESULTS: The gene encoding SgSQS was cloned. SgSQS has 417 amino acid residues with an pI of 7.3. There are 32 phosphorylation sites in its sequence: S48 as well as S196 play important roles in regulation of enzyme activity. The enzyme is a monomeric protein with a cave-like active center formed by α helixes and has two transmembrane domains at its C-terminus. SgSQS mRNA expression in stem and root were about twice as much as that in leaf and peel. Full-length SgSQS with measurable catalytic activity was expressed in Escherichia coli. SgSQS activity was optimal at 37 °C and pH 7.5 respectively. CONCLUSION: SgSQS gene was cloned, and the molecular structure and biochemical function of SgSQS were characterized.

[Establishment of a method to detect duck hepatitis B virus covalently closed circular DNA based on rolling circle amplification].

Rolling circle amplification (RCA) is a newly developed experimental technique that can specific ally amplify circular DNA. Since 2008, RCA has been extensively used in hepatitis B virus (HBV) research, such as the amplification of the full-length sequence of the HBV genome, and the analysis of the drug-resistant mutations of HBV covalently closed circular DNA (cccDNA), amongst others. To create an easy assay for the analysis of duck hepatitis B virus (DHBV) cccDNA, this study established an RCA-based method. DHBV cccDNA was amplified from the DHBV DNA samples of duck liver with four pairs of sulfur-modified primers, which were designed according to the highly conserved sequence of DHBV using sera DHBV DNA as the negative control. DHBV cccDNA was detected in the obtained RCA products by the sequencing of RCA amplicons that were amplified with primer pairs on both sides of the gap of DH BV relaxed circular DNA, rather than by digesting RCA products with a restriction enzyme. The liver and sera DHBV DNA samples of 39 ducks infected with DHBV were examined with the RCA-based DHBV cccDNA detection method, and the results showed that while DHBV cccDNA was detected from all 39 liver DHBV DNA samples, no DHBV cccDNA was found in any of the sera DHBV DNA samples. These results suggest that the method established in the study is highly specific and sensitive for the detection of DHBV cccDNA. The establishment of this RCA-based DHBV method for cccDNA detection lays the groundwork for using a DHBV model to study the role of cccDNA in the pathogenesis of hepatitis B and to evaluate the effect of anti-virus therapies.

A novel complex A/C/G intergenotypic recombinant of hepatitis B virus isolated in southern China.

Hepatitis B virus (HBV) genotypes and subgenotypes may vary in geographical distribution and virological features. Previous investigations, including ours, showed that HBV genotypes B and C were respectively predominant in South and North China, while genotypes A and D were infrequently detected and genotype G was not found. In this study, a novel A/C/G intergenotype was identified in patients with chronic HBV infection in Guilin, a city in southern China. Initial phylogenetic analysis based on the S gene suggested the HBV recombinant to be genotype G. However, extended genotyping based on the entire HBV genome indicated it to be an A/C/G intergenotype with a closer relation to genotype C. Breakpoint analysis using the SIMPLOT program revealed that the recombinant had a recombination with a arrangement of genotypes A, G, A and C fragments. Compared with the HBV recombinants harboring one or two genotype G fragments found in Asian countries, this Guilin recombinant was highly similar to the Vietnam (98-99%) and Long An recombinants (96-99%), but had a relatively low similarity to the Thailand one (89%). Unlike those with the typical genotype G of HBV, the patients with the Guilin recombinant were seropositive for HBeAg. Moreover, a relatively high HBV DNA viral load (>2 × 10(6) IU/ml) was detected in the patients, and the analysis of viral replication capacity showed that the Guilin recombinant strains had a competent replication capacity similar to genotypes B and C strains. These findings can aid in not only the clarification of the phylogenetic origin of the HBV recombinants with the genotype G fragment found in Asian countries, but also the understanding of the virological properties of these complicated HBV recombinants.

[Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV].

Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.

Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B.

Amino acid substitutions at positions rtN238T/D of the hepatitis B virus (HBV) polymerase have been reported as potential mutations associated with adefovir (ADV) resistance. In this study, we characterized the prevalence of the rtN238H mutation and determined the susceptibility to LAM and ADV using phenotypic analyzes in vitro. One thousand eight hundred and sixty-five HBsAg-positive patients with chronic HBV (CHB) infection were included in this study. HBV genotypes and reverse transcriptase (RT) mutations were determined by direct sequencing. Replication-competent HBV constructs containing the naturally occurring rtN238H mutation were generated and replication capacity and susceptibility to LAM and ADV in transiently transfected hepatoma cell lines were determined. Among 1865 enrolled HBV infected patients, 8.8% (165/1865) showed mutations in the rtN238 locus (143 males/22 females, 91 treatment-naive, 42 ADV-treated, 16 LAM-treated and 16 ADV+LAM-treated), namely 86% rtN238H (142/165), 5.5% rtN238S (9/165), 5.5% rtN238T (9/165) and 3% rtN238D (5/165). Among the rtN238H mutant strains, there were no significant differences between ADV- or/and LAM- treated patients and treated-naive patients (42% vs. 58%). Compared with wild-type HBV, this mutant displayed an equivalent susceptibility to LAM or ADV in phenotypic assays. Importantly, we found that the incidence rate of rtN238H was higher in HBV genotype B infected patients than HBV genotype C subsets (80.3% vs. 19.7%), even without exogenous selection pressures. As rtN238H did neither impair the viral replication efficiency nor susceptibility to LAM or ADV in vitro, rtN238H likely represents background polymorphisms rather than resistance mutations with clinical implications. The incidence of rtN238H may be associated with HBV genotype.

[Comparative analysis of drug resistant mutations in the reverse transcriptase domain of hepatitis B virus covalently closed circular DNA and the viral relax circle DNA].

OBJECTIVE: To establish a method for detection of reverse transcriptase region of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to compare the pattern and frequency of drug-resistant mutations in the region between intrahepatic HBV cccDNA and serum HBV relax circle DNA (rcDNA). METHODS: HBV DNA were extracted from liver biopsy tissues of 20 patients with chronic hepatitis B. The RT region of HBV cccDNA was amplified by rolling circle amplification (RCA) followed by polymerase chain reaction (PCR) mediated by a pair of primers spanning across the gap region of HBV genome. The RT region of serum HBV rcDNA from the same patient was amplified by nested-PCR. The PCR products were directly sequenced and analyzed by Vector NTI Suite 8.0 and chromaslite 201 software. x2 test was used for statistical significance analysis of drug-resistant mutation occurrences between the HBV cccDNA and rcDNA. RESULTS: The RT regions of HBV cccDNA were successfully amplified from liver tissues of all enrolled patients using the RCA plus PCR assay. Simultaneously, HBV the RT regions of rcDNA were amplified from these patients serum samples. Sequence analysis showed that the drug-resistant mutations were significantly more frequently detected in HBV rcDNA (40%) than in HBV cccDNA (10%) (P<0.05). Different mutational patterns were observed between the HBV cccDNA and rcDNA in a few cases. CONCLUSION: The RCA in combination with PCR is a practical method for the detection of drug-resistant mutation in the RT region of HBV cccDNA. Drug-resistant mutational patterns could be discrepant between HBV cccDNA and rcDNA.

[Development of a high-efficient assay for amplifying Vbeta-encoding genes of human T-cell receptor].

AIM: To develop an effective assay for amplifying human T-cell receptor (TCR) variable region of beta chain (Vbeta)-encoding genes. METHODS: Based on the property of the 26 subfamilies of human TCR Vbeta-encoding gene sequence, 34 sets of outer and 37 sets of inner sense primers were divided into 8 degenerate primer groups, and a set of outer and inner antisense primers located in conserved region beta chain (Cbeta) was designed for the amplification of the TCR Vbeta-encoding genes. In addition, a sequencing primer and a sense primer for amplifying Cbeta-encoding genes were designed. CD8 T-cell RNA was extracted and subjected to reverse transcription mediated by poly A, followed by a nested polymerase chain reaction (PCR) to amplify the 26 subfamilies of human TCR Vbeta-encoding genes. Jurkat T lymphoma cells were used as control. T-easy vector was employed to detect DNA sequencing of the cloned target genes. RESULTS: All the subfamilies of the Vbeta-encoding genes were obtained from CD8 T cells of a healthy donor, except the subfamily of Vbeta8 , was obtained from Jurkat cells. The results were confirmed by DNA sequencing of the cloned genes. CONCLUSION: The nested RT-PCR assay can effectively amplify human TCR Vbeta-encoding genes with broad spectrum, which is helpful for the cloning and functional study of the TCR expressed by antigen-specific cytotoxic T lymphocytes.

[Analysis and recombinant construction of HBV the reverse transcriptase gene with drug-resistant mutations from 40 patients with chronic hepatitis B].

OBJECTIVE: To analyze genetic mutation associated with drug resistance in the reverse transcriptase (RT) domain of HBV from 40 patients with chronic hepatitis B, and to construct mutant RT gene recombinant vectors for drug-resistant phenotypic analysis. METHODS: HBV DNA was extracted from sera of the 40 patients receiving anti-HBV nucleot (5) ide analogue. The complete RT domain-encoding gene was amplified by nested PCR, and then cloned into pGEM-T-easy vector. Three to Five clones were randomly selected for DNA sequencing. Data were analyzed by UNASTAR software. The pTriEx-HBV (C) 1.1 expression vectors were constructed by replacing the 1250-hp Xho I/Nco I fragments containing complete RT domain from individual patients samples. RESULTS: All samples were detected with drug-resistant mutations associated with lamivudine, adefovir, and entacavir singly or in combination. Ninety-six mutant RT genes were cloned into pGEM-T-easy vector, from which 40 major mutant RT genes were replaced into pTriEx-HBV (C) 1.1 expression vectors. The construction was confinned to be successful by verifying mutation existence using DNA sequencing, and detectable HBsAg and HBeAg in the cell supernatant after transfecting recombinant expression vectors into Huh7 cells. CONCLUSION: The analysis of drug-resistant mutation and the construction of mutant-recombinant expression vectors were successfully implemented using the samples frum clinical patients. The work lays a foundation for drug-resistant phenotypic analysis of HBV mutants.