OBJECTIVEEstablish the fluorescent quantitative RT-PCR detection method for rabies virus (RV) and construct RNase-resistant virus-like particles as positive controls.

METHODSAnalyze the database in GenBank, the probe and the primers were designed in the conservative region of N gene of rabies virus and the method of real-time fluorescent quantitative PCR was obtained; On the basis of MS2 phage, with the technology of gene recombination, prepare the RNase-resistant virus-like particles for RV positive controls;

RESULTSRNase-resistant virus-like particles were obtained after prokaryotic expression in E. coli. The designed primers and probe were confirmed to be very specific and conservative, and be sensitive to-concentration of 15 copies/microl.

CONCLUSIONEstablished the method of detecting rabies virus by reverse transcription real-time quantitative PCR,obtained the RNase-resistant and no infectivity virus-like particles as positive controls of rabies virus.

Animals ; DNA Probes ; DNA, Viral ; Dogs ; Humans ; RNA, Viral ; analysis ; Rabies ; virology ; Rabies virus ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Ribonuclease, Pancreatic ; metabolism ; Viral Load

Objective: To investigate the influence of HBsAg expression in peritumoral tissue of hepatocellular carcinoma (HCC) patients on their postoperative recurrence. Methods: The HCC patients treated in Shanghai Eastern Hepatobiliary Surgery Hospital from October 2009 to August 2010 were selected. The clinicopathological data and adjacent tissues of 718 patients were collected, and dextran polymer immunohistochemical staining was used to detect the expression of HBsAg in adjacent tissues. According to the expression of HBsAg in adjacent tissues, the tissues were divided into HBsAg positive group and HBsAg negative group. Kaplan-Meier method and Log rank test were used for survival analysis, and Cox regression model was used for multivariate analysis. Results: Among the 718 patients in the whole group, 153 were HBsAg negative and 565 were HBsAg positive. There was a statistically significant difference in serum HBV DNA level between HBsAg-positive and HBsAg-negative patients (P<0.001). The number of patients with serum DNA≥2 000 IU/ml and<2 000 IU/ml in HBsAg negative group were 52 and 93, while the patients in HBsAg positive group were 325 and 205. The cumulative recurrence rates of all patients at 1, 3, and 5 years after surgery were 30.2%, 54.3%, and 62.7%, respectively. The expression of HBsAg was related to the recurrence (P=0.038). Multivariate analysis showed that γ-GT, PT, multiple tumors, tumor length, and portal vein invasion were independent risk factors for recurrence of HCC (P<0.05). In HBeAg-negative patients with low viral load (HBV DNA <2 000 IU/ml) and without cirrhosis, the recurrence rates of HBsAg-positive patients were 14.3% and 31.0% at 3 and 5 years, respectively, compared with HBsAg negative patients (all 0), the difference was statistically significant (P=0.021). Conclusion: The positive expression of HBsAg in peritumoral tissue increases the postoperative recurrence risk of HCC patients.
Carcinoma, Hepatocellular/pathology* ; China ; DNA, Viral/analysis* ; Hepatitis B Surface Antigens ; Hepatitis B virus/metabolism* ; Humans ; Liver Neoplasms/pathology*

In order to facilitate the detection of newly emerging or rare viral infectious diseases, a negative-strand RNA virus-severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus-dengue virus, were used to investigate RNA viral genome unspecific amplification by multiple displacement amplification technique from clinical samples. Series of 10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29 DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to 6 X 10(3), even 2 X 10(5) when supplemental RNA existed, and better results were obtained when viral RNA loads were lower. A RNA viral genome amplification system using multiple displacement amplification technique was established in this study and effective amplification of RNA viral genome with low load was achieved, which could provide a tool to synthesize adequate viral genome for multiplex pathogens detection.
Bunyaviridae Infections ; diagnosis ; virology ; Cell Line ; DNA Ligases ; metabolism ; DNA, Complementary ; analysis ; genetics ; DNA-Directed DNA Polymerase ; metabolism ; Dengue ; diagnosis ; virology ; Dengue Virus ; genetics ; isolation & purification ; Genome, Viral ; genetics ; Humans ; Phlebovirus ; genetics ; isolation & purification ; RNA, Viral ; analysis ; genetics ; Reference Standards ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Viral Load

To investigate the genetic stability (including the vector of vaccinia virus and six foreign genes: gp160, gag, pol, rev, tat and nef) of the HIV-1 non-replicating recombinant vaccinia virus (rNTV-C). rNTV-C was serially passaged to passage 25 (P25) in primary chicken embryo fibroblast (CEF). P9, P12, P15 and P25 were selected to study the genetic stability in four aspects, including the genetic stability of viral vector, the genetic stability of six foreign genes, the expressing stability of foreign genes and the genetic loss of foreign genes. The results showed that the viral vector was non-replicated vaccinia virus of Tiantan strain and was passaged stably; foreign gene sequences matched with designed sequences, the insert sites were right, and the nucleotide mutation rate was less than one over ten thousands within different passages of rNTV-C; the target proteins could be expressed effectively, and the expression level was stable within different passages of rNTV-C; the genetic loss of gag and nef was less than 5% within different passages of rNTV-C. The above results provided important data for the vaccine production.
Animals ; DNA, Recombinant ; genetics ; Fibroblasts ; metabolism ; virology ; Gene Expression ; Genes, Viral ; genetics ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; Sequence Analysis, DNA ; Vaccinia virus ; genetics

OBJECTIVETo study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5'UTR.

METHODSPlasmids, 5'UTR-Luc(+) and 5'UTR-Luc(-) carrying the forward and reverse DNA sequences corresponding to the HCV 5'UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5'UTR sequence was cloned into a GFP vector to make 5'UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy.

RESULTS5'UTR-Luc(+) had an obvious luciferase activity whereas 5'UTR-Luc(-) had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the cells transfected with the plasmid of 5'UTR-EGFP.

CONCLUSIONThe forward DNA sequence corresponding to HCV 5'-UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.

5' Untranslated Regions ; genetics ; Carcinoma, Hepatocellular ; virology ; DNA, Viral ; genetics ; Hepacivirus ; genetics ; isolation & purification ; Humans ; Liver Neoplasms ; virology ; Luciferases ; metabolism ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

The relationship between Human papillomavirus (HPV) 16 infection and the natural course of colorectal adenocarcinoma has not been fully defined. In this study, the HPV 16 E7 DNA was detected in 82 patients with primary colorectal adenocarcinoma to study the relationship between HPV 16 infection and colorectal carcinoma. Samples were taken from both the tumors and the adjacent normal mucosa in the same patient. Polymerase chain reaction (PCR) was used to amplify the HPV16 E7 DNA fragment. The PCR products were gel-purified and sequenced for HPV genotyping. DNA sequence analysis indicated that PCR product was 297 bp. It was the equivalent of 562-858th pairs in the HPV 16 primitive sequences. Our results showed HPV16 E7 DNA expression was significantly higher in colorectal carcinoma (42/82) than in adjacent normal mucosa (4/82). The correlation was found between HPV16 E7 expression and tumor's location; the positive rate was 18.18% in the ascending colon carcinoma group and 64.10% in the rectal carcinoma group. High HPV16 E7 expression was also associated with lower Dukes stage (P < 0.01). These results indicated that there was correlation between colorectal adenocarcinoma and HPV 16 infection. HPV16 infection was relatively higher in the colorectal carcinoma and rare in the adjacent normal mucosa. Specimens expressing higher levels of HPV 16 E7 DNA were associated with lower Dukes stage and more distal location.
Adenocarcinoma ; metabolism ; virology ; Adult ; Aged ; Aged, 80 and over ; Colorectal Neoplasms ; metabolism ; virology ; DNA, Viral ; analysis ; Female ; Genotype ; Humans ; Male ; Middle Aged ; Oncogene Proteins, Viral ; biosynthesis ; genetics ; Papillomaviridae ; isolation & purification ; Papillomavirus E7 Proteins ; Papillomavirus Infections ; metabolism ; virology

OBJECTIVETo investigate relationship between glucose metabolic disorders and expression of insulin receptor (IR) and tyrosine protein kinase (TPK) in posthepatitic cirrhosis hepatocyte and HBV DNA expression in pancreatic cells.

METHODSTo detect HBV DNA in paraffin-embedded pancreatic and hepatic tissues from 12 posthepatitic cirrhosis patients with positive serum HBV markers by using in situ hybridization (ISH) with a digoxigenin labelled probe. The amount of IR and TPK have been evaluated by immunohistochemical quantitative analysis using image analyzer in hepatocyte of 12 patients positive for HBV markers with posthepatitic cirrhosis in serum. Immunofluorescent histochemical double staining technique was used. HBsAg and IR were observed under confocal laser scanning microscope.

RESULTSEleven of 12 cirrhosis patients? hepatocytes were HBV DNA positive, including 7 patients (7/7) with impaired glucose tolerance (IGT) and 4 patients (4/5) with normal glucose tolerance (NGT). Eight of 12 pancreatic cells were HBV DNA positive, including 7 patients (7/7) with IGT, but only one patient (1/5) with NGT-HBV DNA was found positive in pancreatic cells in significantly more subjects in IGT group than in NGT group (P less than 0.01).IR and TPK amount in hepatocyte of IGT was significantly less than that of NGT patients with posthepatitic cirrhosis (P less than 0.01). IR amount was closely related to the TPK in cirrhosis hepatocyte r=0.82597(P less than 0.01). HBV DNA was mainly localized in the nuclei of hepatocyte and pancreatic acinar and islet cells. Immunofluorescent histochemical double-staining showed that HBsAg was partly localized in the IR positive areas of hepatocytes and pancreatic islet cells.

CONCLUSIONHBV can invade acinar cells of pancreas and islet cells, which might be a direct cause of insulin-dependent diabetes mellitus-like the disorder and insulin absence after HBV infection. Decrease of IR and TPK might be main cause of noninsulin-dependent diabetes mellitus-like disorder after having hepatitis or posthepatitic cirrhosis.

DNA, Viral ; analysis ; Female ; Glucose Metabolism Disorders ; complications ; metabolism ; virology ; Hepatitis B virus ; genetics ; Hepatocytes ; metabolism ; virology ; Humans ; In Situ Hybridization ; Liver Cirrhosis ; complications ; metabolism ; virology ; Male ; Middle Aged ; Pancreas ; cytology ; virology ; Protein-Tyrosine Kinases ; metabolism ; Receptor, Insulin ; metabolism

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

OBJECTIVEBy studying the possibility of obtaining expression of human hepatitis B virus (HBV) genes and production in normal liver cells from heterologous species like normal primary duck hepatocytes (PDH), to investigate the species-specificity of HBV infection and replication.

METHODSTwo days after transfecting the complete HBV genome into PDH by electroporation (transfected group), HBsAg and HBeAg in the supernatants and lysates of PDH were measured by the IMX system. Meanwhile, replication of HBV in PDH was analyzed by Southern blotting and dot blotting procedures. PDH was electroporated as control.

RESULTSHBsAg in the lysate of transfected group was 9.10 (P/N values, positive?2.1), HBeAg was 1.0 (negative?2.1), both were negative in the supernatants of transfected group. dot blotting revealed that transfected group was strongly positive, whereas the control group was negative. Southern blot analysis of intracellular total DNA indicated that there were relaxed circular (RC), covalently closed circular (CCC) and single-stranded (SS) HBV DNA replicative intermediates in the transfected group, and there was no integrated HBV DNA in the cellular genome. Control groups were negative.

CONCLUSIONSReplication of HBV can occur in hepatocytes from nonmammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Animals ; Cells, Cultured ; DNA Replication ; physiology ; DNA, Viral ; biosynthesis ; Disease Models, Animal ; Ducks ; Electroporation ; Hepatitis B Surface Antigens ; analysis ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; metabolism ; virology ; Humans ; Species Specificity ; Transfection ; Virus Replication

OBJECTIVETo clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein.

METHODSUsing the yeast dual hybrid system 3, bait plasmids of hepatitis C virus core were constructed. After identifying hepatitis C virus core protein that could stably expressed in AH109 yeast strains, we performed yeast two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for alpha-gal activity. The genes of yeast colonies that could grow on QDO and had alpha-gal activity were sequenced.

RESULTSAmong the 30 positive colonies, we blasted the gene of the sixth colony; we coined human hepatitis C virus binding protein 6(Hu Hcbp6) with Genbank, realized that the Hu Hcbp6 shares as much as 98% homology with two cDNA without knowing functions. We have proved that Hu Hcbp6 could interact with hepatitis C virus core protein.

CONCLUSIONSHepatitis C virus core binding protein (Hu Hcbp 6 Genbank number: AY032594) was successfully cloned and identified. The study partly paved the way for investigating physiological function of the Hu Hcbp6.

Cloning, Molecular ; DNA, Complementary ; genetics ; DNA-Binding Proteins ; genetics ; Hepacivirus ; Humans ; Molecular Sequence Data ; Plasmids ; Sequence Analysis, DNA ; Transfection ; Two-Hybrid System Techniques ; Viral Core Proteins ; genetics ; metabolism ; Yeasts ; genetics