OBJECTIVE: To determine the expression profile of microRNA (miRNA) in peripheral blood mononuclear cells (PBMC) and immune factors in pregnant women with hepatitis B virus (HBV) infection. METHODS: A total of 182 pregnant women infected with HBV were randomly selected, with 40 healthy pregnant women and 35 non-pregnant women as controls. High-throughput sequencing was used to detect RNA in the PBMC of all subjects. Indirect ELISA method was used to determine the changes of cytokines in peripheral blood samples. RESULTS: Compared with the control group, 18 differentially expressed miRNA were identified in those with HBV infection (P< 0.01). Among these, miR-3607-3p, miR-20a, miR-1296, miR-153-1 and miR-X4 may directly regulate the transcriptional level of target genes including IL-10, IL-18, IL-16, MCP-1, NUP50 and CCR1. Meanwhile, peripheral blood cytokines IL-10, IL-18, IL-16 and MCP-1 were significantly increased in those with HBV infection (P<0.01), with the expression level of IL-16 and MCP-1 being strongly correlated with the viral load. CONCLUSION The expression profiles of miRNA in PBMC and cytokines in peripheral blood can change significantly during pregnancy, both may be involved in the immune response to HBV infection.
Cytokines ; blood ; DNA, Viral ; Female ; Hepatitis B ; blood ; Hepatitis B virus ; Humans ; Leukocytes, Mononuclear ; metabolism ; MicroRNAs ; blood ; Pregnancy

OBJECTIVESIn order to investigate the relationship among the toll-like receptor 2 (TLR2), hepatitis B e antigen and HBV DNA, the expression levels of TLR2 on peripheral blood monocytes of chronic hepatitis B (CHB) patients as well as on their monocytes stimulated by ligand of TLR2 (Pam3CSK4) and HBeAg were analyzed.

METHODSSixty-eight adults with CHB were enrolled, including 37 HBeAg-positive patients, 17 HBeAg-negative and HBV DNA negative patients, and 14 HBeAg-negative and HBV DNA positive patients. Sixteen healthy volunteers were also studied as controls. TLR2 expression levels on their peripheral blood monocytes stimulated with Pam3CSK4 or not stimulated were analyzed by FACS Caliber. The relationship of the expression levels of TLR2, HBeAg and HBV DNA were also analyzed. The level of TLR2 on peripheral blood monocytes of healthy volunteers and HBeAg-negative CHB patients stimulated by HBeAg was examined for six hours.

RESULTSThe TLR2 expression levels on CD14+ cells were significantly reduced in HBeAg-positive patients (47.57%+/-21.40 %) compared to both healthy volunteers (76.51%+/-7.46%) and HBeAg-negative patients (HBV DNA positive group 73.2%+/-14.2%, HBV DNA negative group 75.2%+/-11.3%); but there was no difference between those of the HBeAg-negative patients and the healthy volunteers. Expression levels of TLR2 on monocytes stimulated by TLR2 ligand in HBeAg-positive patients were obviously increased, and reached the basic levels of the healthy volunteers and the HBeAg-negative patients. The expression levels of TLR2 on monocytes stimulated by HBeAg of the healthy volunteers and the HBeAg-negative patients were markedly reduced.

CONCLUSIONSIn the presence of HBeAg, HBV down-regulates the expressions of TLR2 on CD14+ cells from peripheral blood, and there is no correlation between HBV-DNA and TLR2. Pam3CSK4 can boost the TLR2 expression in HBeAg-positive patients. The proposed interaction between HBV and TLR2 may provide an important clue to explain the reasons of the establishment of persistent HBV infection.

Case-Control Studies ; DNA, Viral ; blood ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; blood ; immunology ; metabolism ; Humans ; Lipopolysaccharide Receptors ; metabolism ; Monocytes ; metabolism ; Toll-Like Receptor 2 ; metabolism

OBJECTIVETo investigate the changes in serum hepatitis B viral load (HBV DNA) and transformer growth factor-β(1)(TGF-β1) in patients with chronic hepatitis B after entecavir treatment and evaluates the therapeutic effect of entecavir.

METHODSSixty-three patients with chronic hepatitis B were randomly assigned into entecavir group (n=33) and control group (n=30). The entecavir group consisted of 9 mild, 17 moderate, and 7 severe cases, all treated with oral entecavir (0.5 mg daily) and general hepatoprotective drugs; the control group, consisting of 13 mild, 12 moderate and 5 severe cases, was treated with the hepatoprotective drugs only. Serum HBV DNA and TGF-β(1)were determined before and at 3 and 6 months during the treatment.

RESULTSEntecavir treatment reduced serum HBV DNA load in all the cases in entecavir group, and the difference was statistically significant between the levels measured at 3 and 6 months (P<0.05). The treatment also resulted in decreased serum TGF-β(1)levels in moderate and severe cases, and the severe cases showed a significant TGF-β(1)reduction after a 6-month treatment (P<0.05).

CONCLUSIONEntecavir can lower serum HBV DNA load levels in patients with chronic hepatitis B. Entecavir is also effective to reduce serum TGF-β(1)levels in moderate and severe cases, especially in the latter.

Adult ; Aged ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Guanine ; analogs & derivatives ; therapeutic use ; Hepatitis B virus ; drug effects ; Hepatitis B, Chronic ; blood ; drug therapy ; virology ; Humans ; Middle Aged ; Serum ; metabolism ; Transforming Growth Factor beta1 ; blood ; Viral Load ; Young Adult

Occult HBV infection is characterized by the absence of serum HBsAg with persistence of low level of intrahepatic HBV DNA. Several suggested mechanisms for the origin of occult HBV infection include strong suppression of viral replication and gene expression, mutation in the regulatory regions of HBV genome, formation of immunoglobulin-bound HBsAg, viral interference, and blockage of HBsAg secretion from infected hepatocytes. Standardized assays are not yet available, and sensitive HBV DNA amplification assay is necessary for the diagnosis of cryptic infection. Detection rate of HBV DNA is highest in IgG anti-HBc positive population. However, neither anti-HBc nor anti-HBs can be detected in a significant proportion of infected persons. Occult HBV infection occurs in a number of clinical settings and is highly prevalent in HCV-infected patients as well as in patients with cryptogenic chronic liver disease including hepatocellular carcinoma.
DNA, Viral/analysis ; Hepatitis B/*diagnosis/*epidemiology/metabolism ; Hepatitis B Antibodies/blood ; Hepatitis B Core Antigens/immunology ; Hepatitis B Surface Antigens/blood ; Humans

OBJECTIVEWe have evaluated the direct effect of ampelopsis (APS) on duck hepatitis B virus (DHBV) replication in ducklings in vivo.

METHODOne-day-old ducklings were infected with DHBV. After infection for 7 days, the animals were treated with APS at dosages of 70, 150, 300 mg x kg(-1) of body weight via the oral route. The drug was given twice per day for 10 days continuously, and normal saline was used as control. The blood was drawn from the posterior tibial vein of all ducks before treatment (T0), after the medication for 5 (T5), 10 (T10) days and withdrawal of the drug for 3 days (P3). DHBV DNA in duck serum was detected by dot blot.

RESULTThe duck serum DHBV-DNA levels were reduced in the group of APS (150, 300 mg x kg(-1)) after treated for 5 and 10 days and the levels of DHBV-DNA did not markedly relapse in both groups of APS after withdrawal of the drug for 3 days. We provide the first evidence that APS can efficiently inhibits DHBV replication in ducks in vivo.

CONCLUSIONAPS therefore warrants further investigation as a potential therapeutic agent for HBV infections.

Ampelopsis ; chemistry ; Animals ; Antiviral Agents ; pharmacology ; DNA, Viral ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ducks ; blood ; virology ; Hepatitis B Virus, Duck ; drug effects ; metabolism ; physiology ; Virus Replication ; drug effects

BACKGROUND: Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. METHOD: 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. RESULTS: The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. CONCLUSIONS: Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients.
Antiviral Agents/therapeutic use ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/drug therapy/pathology/virology ; DNA, Viral/*blood/metabolism ; Ganciclovir/therapeutic use ; Humans ; *Immunoassay ; Organ Transplantation ; Phosphoproteins/genetics/immunology/*metabolism ; *Real-Time Polymerase Chain Reaction ; Viral Matrix Proteins/genetics/immunology/*metabolism ; Virology/*methods

OBJECTIVETo investigate the expressions of perforin (PF), granzyme B (GrB), granulysin (GNLY), TNF-alpha and IFN-gamma in peripheral CD8+ T lymphocytes and their correlation to infection status in patients with chronic hepatitis B (CHB).

METHODSALT, AST, TB and HBV DNA copy were detected to evaluate the infection status in CHB patients, with healthy volunteers serving as the control group. According to the infection status, the CHB patients were divided into 4 groups, namely normal hepatic function and high HBV DNA level group, normal hepatic function and low HBV DNA level group, abnormal hepatic function and high HBV DNA level group and abnormal hepatic function and low HBV DNA level group. The expressions of some immune effector molecules in CD8+T cells were detected by flow cytometry, and the correlations between these immune effector molecules and the infection status were analyzed.

RESULTSThe expressions of GrB, TNF-alpha and IFN-gamma in normal hepatic function and low HBV DNA level group were significantly higher than those in abnormal hepatic function and high HBV DNA level group (P<0.05). The expression of IFN-gamma in normal hepatic function and high HBV DNA level group was significantly higher than that in abnormal hepatic function and high HBV DNA level group (P<0.05). The expressions of PF and GNLY were similar among all the 4 groups. Positive correlations were noted between GrB, PF, GNLY, TNF-alpha and IFN-gamma.

CONCLUSIONGrB, TNF-alpha and IFN-gamma in peripheral CD8+ T cells are inversely correlated to hepatic dysfunction and HBV DNA level in CHB patients.

Adult ; CD8-Positive T-Lymphocytes ; immunology ; metabolism ; Case-Control Studies ; DNA, Viral ; blood ; Female ; Granzymes ; blood ; Hepatitis B, Chronic ; blood ; immunology ; Humans ; Interferon-gamma ; blood ; Liver ; physiopathology ; virology ; Male ; Middle Aged ; Perforin ; blood ; Tumor Necrosis Factor-alpha ; blood ; Young Adult

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

Adolescent ; Adult ; Aged ; Cells, Cultured ; DNA, Viral ; blood ; Female ; Flow Cytometry ; Hepatitis B Core Antigens ; blood ; Hepatitis B Surface Antigens ; blood ; Hepatitis B, Chronic ; metabolism ; virology ; Humans ; Interferon-alpha ; blood ; metabolism ; Interleukin-18 ; blood ; metabolism ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Male ; Middle Aged ; Oligodeoxyribonucleotides ; pharmacology ; Polymerase Chain Reaction ; methods ; Toll-Like Receptor 9 ; agonists ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Young Adult

Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10(6) IU/mL (R2 ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values <1,000 copies/mL, 100% of the samples had a variation <0.7 log10 copies/mL; >1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had <0.5 log10 copies/mL. The Real-Q assay is useful for quantifying CMV in WB with the two real-time PCR platforms.
Cytomegalovirus/*genetics/isolation & purification ; Cytomegalovirus Infections/diagnosis/virology ; DNA, Viral/*blood/metabolism ; Humans ; Limit of Detection ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction