Objective: To understand the distribution of genotypes and sub-genotypes of HBV in different ethnic groups in China. Methods: The HBsAg positive samples were selected by stratified multi-stage cluster sampling from the sample base of national HBV sero-epidemiological survey in 2020 for the amplification of S gene of HBV by nested PCR. A phylogeny tree was constructed to determine the genotypes and sub-genotypes of HBV. The distribution of genotypes and sub-genotypes of HBV were analyzed comprehensively by using laboratory data and demographic data. Results: A total of 1 539 positive samples from 15 ethnic groups were successfully amplified and analyzed, and 5 genotypes (B, C, D, I and C/D) were detected. The proportion of genotype B was higher in ethnic group of Han (74.52%, 623/836), Zhuang (49.28%, 34/69), Yi (53.19%, 25/47), Miao (94.12%, 32/34), Buyi (81.48%, 22/27). The proportions of genotype C were higher in ethnic groups of Yao (70.91%, 39/55). Genotype D was the predominant genotype in Uygur (83.78%, 31/37). Genotype C/D were detected in Tibetan (92.35%,326/353). In this study, 11 cases of genotype I were detected, 8 of which were distributed in Zhuang nationality. Except for Tibetan, sub-genotype B2 accounted for more than 80.00% in genotype B in all ethnic groups. The proportions of sub-genotype C2 were higher in 8 ethnic groups, i.e. Han, Tibetan, Yi, Uygur, Mongolian, Manchu, Hui and Miao. The proportions of sub-genotype C5 were higher in ethnic groups of Zhuang (55.56%, 15/27) and Yao (84.62%, 33/39). For genotype D, sub-genotype D3 was detected in Yi ethnic group and sub-genotype D1 was detected in both Uygur and Kazak. The proportions of sub-genotype C/D1 and C/D2 in Tibetan were 43.06% (152/353) and 49.29% (174/353). For all the 11 cases of genotype I infection, only sub-genotype I1 was detected. Conclusions: Five genotypes and 15 sub-genotypes of HBV were found in 15 ethnic groups. There were significant differences in the distribution of genotypes and sub-genotypes of HBV among different ethnic groups.
Humans ; Asian People ; China/epidemiology* ; Ethnicity ; Genotype ; Gerbillinae ; Hepatitis B virus/genetics* ; Hepatitis B/virology*

OBJECTIVE: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. METHODS: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. RESULTS: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. CONCLUSIONS The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
Computational Biology ; DNA Methylation ; DNA, Circular/genetics* ; DNA, Viral/genetics* ; Genome, Human ; Genomics ; Genotype ; Hepacivirus/genetics* ; Hepatitis C/virology* ; Humans ; Leukocytes, Mononuclear/virology* ; Polymerase Chain Reaction ; RNA, Viral/genetics* ; Sequence Alignment

OBJECTIVE: To explore the diagnostic value of the serum metabolites identified by high-performance liquid chromatography-mass spectrometry (HPLC/MS) for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). METHODS: A total of 126 patients admitted to Tianjin Third Central Hospital were enrolled, including 27 patients with HBV-related hepatitis with negative viral DNA (DNA-N), 24 with HBV-related hepatitis with positive viral DNA, 24 with HBV-related liver cirrhosis, 27 with HBV-related HCC undergoing surgeries or radiofrequency ablation, and 24 with HBV-related HCC receiving interventional therapy, with 25 healthy volunteers as the normal control group. Serum samples were collected from all the subjects for HPLC/MS analysis, and the data were pretreated to establish an orthogonal partial least- squares discriminant analysis (OPLS-DA) model. The differential serum metabolites were preliminarily screened by comparisons between the HBV groups and the control group, and the characteristic metabolites were identified according to the results of non-parametric test. The potential clinical values of these characteristic metabolites were evaluated using receiver operator characteristic curve (ROC) analysis. RESULTS: A total of 25 characteristic metabolites were identified in the HBV- infected patients, including 9 lysophosphatidylcholines, 2 fatty acids, 17α-estradiol, sphinganine, 5-methylcytidine, vitamin K2, lysophosphatidic acid, glycocholic acid and 8 metabolites with few reports. The patients with HBV- related HCC showed 22 differential serum metabolites compared with the control group, 4 differential metabolites compared with patients with HBV-related liver cirrhosis; 10 differential metabolites were identified in patients with HBV-related HCC receiving interventional therapy compared with those receiving surgical resection or radiofrequency ablation. From the normal control group to HBV-related HCC treated by interventional therapy, many metabolites underwent variations following a similar pattern. CONCLUSIONS We identified 25 characteristic metabolites in patients with HBV-related HCC, and these metabolites may have potential clinical values in the diagnosis of HBV-related HCC. The continuous change of some of these metabolites may indicate the possibility of tumorigenesis, and some may also have indications for the choice of surgical approach.
Carcinoma, Hepatocellular ; blood ; diagnosis ; virology ; Case-Control Studies ; Chromatography, High Pressure Liquid ; DNA, Viral ; blood ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; blood ; virology ; Humans ; Liver Cirrhosis ; virology ; Liver Neoplasms ; blood ; diagnosis ; virology ; Mass Spectrometry ; Metabolome ; Metabolomics ; ROC Curve

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

Objective: To investigate the effects of hepatitis B virus (HBV) genotype and mutations on the development of hepatocellular carcinoma (HCC) and to establish a new qualified HCC risk scores. Methods: A cohort study enrolling patients with chronic HBV infection was conducted. HBV genotypes were identified by nested multiplex PCR. HBV mutations in the basic core promoter region and PreS region were sequenced after PCR amplification. Scores on risk factors were set based on nomogram. Results: Totally, 1 525 patients were followed-up in this research. A total of 1 110 patients infected with genotype C were followed-up for 8.52 (Q(R): 5.36-11.68) years on average, of whom the incidence of HCC was 11.93/1 000 person-years. In genotype C HBV infected patients, male gender, aged 40 years and over, and four DNA mutations (T1674CG, A1762T/G1764A, A3120T, and A2962G) can increase the risk of HCC (P<0.05); interferon therapy can reduce the risk of HCC (P<0.05). A new HCC predicting model was established according to the results. After validation, the predicted disease-free survival rate was consistent with the real one. Conclusions: Hepatitis B virus genotypes and mutations were closely associated with HCC. The new risk scoring system can well predict HCC occurrence in genotype C HBV infected patients.
Adult ; Aged ; Carcinoma, Hepatocellular/virology* ; China/epidemiology* ; Cohort Studies ; DNA, Viral/genetics* ; Female ; Genotype ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/complications* ; Humans ; Liver Neoplasms/virology* ; Male ; Middle Aged ; Mutation ; Predictive Value of Tests ; Risk Factors ; Sensitivity and Specificity

Objective: To explore the SNP effects of patatin-like phospholipase domain which containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2) gene, environmental effects of smoking, alcohol drinking and interaction between gene-gene, gene-environment and drinking-smoking on hepatitis B virus-associated hepatocellular carcinoma (HBV-HCC). Methods: We collected anticoagulant peripheral blood from patients of HBV-HCC, chronic hepatitis B (CHB), liver cirrhosis (LC) and from healthy controls to detect the single nucleotide polymorphism (SNP) of patatin-like phospholipase domain containing 3 (PNPLA3) gene loci rs738409 and transmembrane 6 superfamily member 2 (TM6SF2) gene loci rs58542926, using the flight mass spectrometry method. The optimal assignment value of gene polymorphisms was defined by using the online SNP stats. Hardy-Weinberg (H-W) balance was tested for SNP. Effects of the genetic and environmental factors to HBV-HCC were analyzed by using the multiple classification logistic regression method. The gene-gene, gene-smoking and alcohol drinking interaction effects were investigated by Fork-Life analysis and binary logistic regression methods. Results: The frequency distribution of CHB group rs738409 loci seemed not in conformity with the H-W balance (χ(2)=11.980, P<0.005). Two loci frequency distributions in the other groups were all in accordandce with the H-W balance. After adjusting for influences on age and sex and comparing to the healthy group, the rs58542926 mutation appeared as OR=1.659, 95%CI: 1.026-2.684, P=0.039, in the HBV-HCC group. When comparing to CHB group, the HBV-HCC group presented that drinking as OR=1.680, 95%CI: 1.121-2.519, P=0.012. When comparing to the LC group, the ORs of drinking and smoking were 1.539 (1.071-2.213) and 1.453 (1.005-2.099) respectively, in the HBV-HCC group. When comparing to the CHB+LC group, interactions between the HBV-HCC group were found rs738409 and rs58542926 on additive model OR=1.548 (U=1.885, P=0.029) and OR=1.658 (P=0.024) on logistic regression model while drinking was rs738409 on interaction additive model with OR=1.811(U=1.965, P=0.024). As for drinking and mutation of rs738409, the multiplication model of logistic regression showed no statistically significant differences. Interaction between smoking and drinking appeared as OR=1.756 (P<0.001) in the logistics regression multiplication model. Conclusions: Factors as mutation of TM6SF2, smoking and drinking all appeared as risk factors for HBV-HCC. Mutations of both PNPLA3 and TM6SF2, together with smoking and drinking all served as risk factors for HBV-HCC. However, the mutation of single PNPLA3 appeared as a protective factor on HBV-HCC.
Alcohol Drinking/adverse effects* ; Carcinoma, Hepatocellular/virology* ; Case-Control Studies ; Epistasis, Genetic ; Gene-Environment Interaction ; Genetic Predisposition to Disease ; Genotype ; Hepatitis B virus ; Hepatitis B, Chronic ; Humans ; Lipase/genetics* ; Liver Cirrhosis, Alcoholic/complications* ; Liver Neoplasms/virology* ; Membrane Proteins/genetics* ; Polymorphism, Single Nucleotide ; Smoking/adverse effects*

OBJECTIVEHepatitis Delt a Virus (HDV) antigen is widely used as a capture antigen in ELISAs for the identification of HDV infection; large amounts of recombinant HDV antigen with active antigenicity are required for this purpose.

METHODSReconstruct the gene of HDV antigen based on the bias code of Escherichia coli, the recombinant protein expresses by high-density fermentation with fed-batch feeding strategy, and purify by immobilized metal chromatography. The sensitivity and specificity of this antigen detect by ELISA method.

RESULTSThe expression of HDV antigen can reach 20% of the total cell mass in the soluble form. The recombinant HDV antigen can be conveniently purified (98%) by immobilized metal ion affinity chromatography (IMAC) using the interaction between a His-tag and nickel ions. Production of recombinant HDV antigen can reach 0.5 g/L under conditions of high-density cell fermentation. Applied to the diagnostic ELISA method, the recombinant HDV antigen shows excellent sensitivity (97% for IgM and 100% for IgG) and specificity (100% for IgG and IgM) for the detection of anti-HDV antibodies.

CONCLUSIONExpression and purification the recombinant HDV antigen as a candidate protein for application in a diagnostic ELISA for HDV infection. Large-scale production of the protein can be achieved using the high-density fermentation strategy.

Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Hepatitis D ; diagnosis ; immunology ; virology ; Hepatitis Delta Virus ; immunology ; Hepatitis delta Antigens ; immunology ; Recombinant Proteins ; genetics ; metabolism

Bone marrow stromal antigen 2 (BST-2) is a kind of host restriction factor. Since it was discovered to be responsible for the defect in virion release of HIV-1 mutants lacking the accessory gene vpu in 2008, it was thought to mainly restrict the viruses by directly tethering viral particles at the plasma membrane. Recent reports suggest that BST-2 also can inhibit the the release of HBV particles, which are budding in the intracellular vesicles, expanding the antiviral spectrum of BST-2. Futhermore, the machanism that BST-2 used to restrict HBV release in multivesicular bodies (MVBs) is similar to that used to restrict HIV at the plasma membrane. However, HBV have evolved strategies to antagonize the antiviral action of BST-2. There are two different opinions about the antagonist. One is HBV inactivated BST-2 by HBx requiring a hepatocyte-specific environment. Another thought envelope protein HBs counteract the antiviral action of BST-2. In this review, we focus on the current advances in the anti-HBV activity of BST-2.
Animals ; Antigens, CD ; genetics ; immunology ; GPI-Linked Proteins ; genetics ; immunology ; Hepatitis B ; genetics ; immunology ; virology ; Hepatitis B virus ; genetics ; physiology ; Host-Pathogen Interactions ; Humans ; Virus Release

We wished to undertake molecular genetic typing and evaluate recombinants of the hepatitis-B virus (HBV) in Tibet (China). Multistage random sampling was used to collect HBsAg-positive samples. Nested polymerase chain reactions were used to amplify the whole sequence of the HBV. DNAstar, MEGA6 and SimPlot were used to assemble sequences, create phylogenetic trees, and undertake recombination analyses. Twelve whole sequences of the HBV of a Tibetan population were collected using these methods. Results showed that all 12 strains were C/D recombinants. Nine of the recombinations were at nt750, and the other three at nt1526. Therefore, the 12 strains could be divided into two types of recombinants: C/Da and C/Db. Analyses of the sequence of the whole genome revealed that the 12 strains belonged to genotype C, and that the nucleotide distance was > 4% between the 12 strains and sub-genotypes C1 to C15 in Genbank. The most likely sub-genotype was C1. Individuals with C/Da were from central and northern Tibet (e.g., Lasa, Linzhi, Ali) and those with C/Db recombinants were from Shannan in southern Tibet. These data suggest that the two types of recombinants had a good distribution in Tibet. Also, they can provide important information for studies on HBV recombination, gene features, virus evolution, as well as the control and prevention of HBV infection in Tibet.
Adult ; Female ; Genotype ; Hepatitis B ; virology ; Hepatitis B virus ; classification ; genetics ; isolation & purification ; Humans ; Male ; Phylogeny ; Recombination, Genetic ; Sequence Analysis, DNA ; Tibet ; Young Adult

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.
Animals ; Cell Line, Tumor ; Cloning, Molecular ; DEAD Box Protein 58 ; antagonists & inhibitors ; genetics ; immunology ; Fibroblasts ; immunology ; pathology ; Gene Expression ; Hepatitis B ; genetics ; immunology ; pathology ; veterinary ; Hepatitis B Virus, Woodchuck ; Immunity, Innate ; Interferon-beta ; genetics ; immunology ; Isoelectric Point ; Kidney ; immunology ; pathology ; virology ; Liver ; immunology ; pathology ; virology ; Marmota ; genetics ; immunology ; virology ; Open Reading Frames ; Protein Domains ; RNA, Double-Stranded ; RNA, Small Interfering ; genetics ; metabolism ; Rodent Diseases ; genetics ; immunology ; pathology ; virology