Antigens, Viral ; genetics ; metabolism ; Cell Line ; Cell Phone ; Gene Expression Regulation, Viral ; radiation effects ; Herpesvirus 4, Human ; physiology ; radiation effects ; Humans ; Immunohistochemistry ; Viral Proteins ; genetics ; metabolism

OBJECTIVETo study the relativity between La protein and the stability of HBV mRNA and the expression of HBV protein.

METHODSFour specific siRNAs were obtained by transcription in vitro. After transfection with the siRNAs into HepG2.2.15 cells for 3 days, the inhibitive effects of La protein were analyzed by Western blot; the content changes of HBsAg, HBeAg and HBV-DNA were detected by ECL and RT-PCR.

RESULTSIn comparison to normal cells, La protein was less in the cells. There was less La protein in the cells trans-infected with siRNAs. HBsAg, the HBeAg and HBV-DNA secreted by the cells transfected with siRNA were also less than that in the normal cells.

CONCLUSIONThere is a correlation between La protein and HBV mRNA and the expression of HBV protein.

Autoantigens ; metabolism ; Cell Line, Tumor ; DNA, Viral ; Hepatitis B Surface Antigens ; Hepatitis B virus ; genetics ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; RNA, Viral ; Ribonucleoproteins ; metabolism

OBJECTIVETo investigate the effect of miR-122 on the expression of hepatitis B virus (HBV) proteins.

METHODSAnti-sense oligodeoxynucleotide (ASODN) of two different sequences against miR-122, anti-miR-122 and LNA-antimiR-122 (Locked nucleic acid), human miR -122 (hsa-miR-122), or the negative control anti-GFP were designed and synthesized, then transfected into HepG2.2.15 cells. After 24 h and 48 h, the levels of HBsAg and HBeAg in the supernatant were determined with a time-resolved immunofluorometric assay (TRFIA). HBV DNA in supernatant and miR-122 in cells were measured by quantitative real-time PCR.

RESULTSAfter 48 h expressions of miR-122 in the LNA-antimiR-122 and anti-miR-122 groups were significantly suppressed and lower than those in the negative control (P<0.001), while the level of miR-122 in the hsa-miR-122 group was higher than that in the negative control (P<0.001). The expression of HBeAg and HBsAg in hsa-miR-122 group was lower than that in the negative control (P<0.01) 24 h and 48 h after transfection. The expression of HBeAg and HBsAg in the anti-miR-122 group and LNA-antimiR-122 group was significantly lower than that in negative control (P>0.001). The levels of viral DNA at both time-points in the various test groups were not significantly different from those of negative control group (P>0.05).

CONCLUSIONmiR-122 may regulate HBV antigens and potentially affect the progress of pathogenesis, which might be the new targets for treatment of HBV infection.

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; MicroRNAs ; genetics ; metabolism ; Transfection

Antigens, Viral ; metabolism ; China ; epidemiology ; Humans ; Influenza B virus ; classification ; genetics ; immunology ; Phylogeny

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3Dpol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3Dpol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp26-->Glu substitution in a beta sheet located within a small groove of the 3Dpol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.
Amino Acid Sequence ; Amino Acid Substitution ; Antigens, Viral/chemistry/*genetics/metabolism ; Capsid Proteins/chemistry/*genetics/metabolism ; Cloning, Molecular ; Foot-and-Mouth Disease Virus/classification/*genetics/*metabolism ; Gene Expression Regulation, Viral ; Molecular Sequence Data ; Phylogeny ; Viral Nonstructural Proteins/chemistry/*genetics/metabolism

In order to increase the expression level of target gene and to simplify the purifying process of separation and purification, we performed the transgenetic research of antigen VP7 gene into peanut via Agrobacterium tumefaciens. The plant binary expression vector is pBOG3VP7 harboring fusion gene oleosin-vp7, which is promoted by ole-promoter. Cotyledon nodes were used as transformation recipients. Transformed individuals were obtained through selection on medium containing 125 mg L-1 Kan. Integration of transgenes was assessed by PCR amplification and PCR-Southern blot hybridization. Taking pBOG3VP7 plasmid as positive control, non-transformed peanut as negative control. 6 plants among 11 plants grown up through seletion medium were detected by PCR and the rate of positive plants is 54.5%. PCR positive plants were further analysed by PCR-Southern blot hybridization. The results showed that 3 plants have DNA bloting bands. The results also showed that the foreign gene was integrated into genome of transformed peanuts. Elevated expression of rotavirus VP7 antigen in transgenic peanuts was a critical factor in the development of efficient and cheap plant oral vaccine.
Agrobacterium tumefaciens ; genetics ; Antigens, Viral ; biosynthesis ; genetics ; Arachis ; genetics ; metabolism ; Capsid Proteins ; biosynthesis ; genetics ; Plants, Genetically Modified ; genetics ; metabolism ; Rotavirus ; genetics ; immunology ; Transformation, Genetic ; Vaccines, Synthetic

OBJECTIVESTo evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro.

METHODS(1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry.

RESULTSOur data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The expression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment.

CONCLUSIONOur results demonstrated that siRNAs exerted robust and specific inhibition on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; DNA, Viral ; biosynthesis ; Flow Cytometry ; Gene Expression Regulation, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; RNA, Small Interfering ; genetics ; metabolism ; Virus Replication ; genetics

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.
Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Antigens, Viral ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; CpG Islands ; genetics ; Cytomegalovirus ; immunology ; DNA ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Lectins, C-Type ; Lentivirus ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism

OBJECTIVETo establish a cell model with HBV secretion by plasmid transfection with whole HBV genes into hepatic L02 cells, and to analyze the effect of HBV on the expression of HLA-A, B, C and MICA/B in L02 cells.

METHODSThe mock control plasmid was built by digesting pEcob6 plasmid with EcoR I at the HBV DNA site and ligating the fragment without HBV DNA. The L02 cells were transfected with pEcob6 or mock plasmid and pcDNA3 1-neo by liposome. The expressions of HBsAg, HBcAg/HBeAg and HBV DNA were detected by immunofluorescence assay, Abbott enzymoimmunoassay, or FQ-PCR. The expressions of HLA-A, B, C and MICA/B were determined by FACS and the differences in the two molecules were analyzed.

RESULTSAfter the transfected cells were selected by G418, the HBsAg and HBeAg in the supernatant of L02-HBV cells were 24.78(S/N) and 4.117(S/N). The quantity of HBV DNA was 9.67 x 10(4) copies/ml, but in L02-mock and in L02 cells they were all negative. Under a confocal microscope HBsAg was brightly shown in the cytoplasm while HBcAg showed dimly in the cytoplasm or in the nuclei. By using FACS, the L02 and L02-mock cells showed some low expressions of HLA-A, B, C and a little expression of MICA/B, while the expression of the two molecules was higher in L02-HBV and the differences were significant (P < 0.05).

CONCLUSIONA cell model with the expression of HBV antigens and the secretion of HBV DNA was established by pEcob6 transfection into L02 cells, the transcription or replication of HBV gene might induce stronger expressions of HLA-A, B, C and MICA/B on hepatic cells. They might be related to the immune injuries of hepatic cells.

Cell Line ; DNA, Viral ; secretion ; Gene Expression ; Gene Expression Regulation, Viral ; Genetic Vectors ; HLA Antigens ; genetics ; Hepatitis B Antigens ; genetics ; metabolism ; Hepatitis B virus ; genetics ; Histocompatibility Antigens Class I ; genetics ; Humans ; Transfection

Hepatitis C virus (HCV) is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.
Antigens, CD81/metabolism ; Genome, Viral ; Hepacivirus/genetics/*physiology ; Humans ; RNA, Viral/metabolism ; Scavenger Receptors, Class B/metabolism ; Viral Envelope Proteins/chemistry/metabolism ; Viral Nonstructural Proteins/chemistry/metabolism ; Virus Assembly ; Virus Internalization ; Virus Replication