Carcinoma, Hepatocellular ; virology ; Hepatitis B virus ; genetics ; pathogenicity ; Humans ; Liver Neoplasms ; virology ; Receptors, Virus ; metabolism ; Virus Integration

OBJECTIVETo compare the performance of Inverse-PCR, Alu-PCR and Cassette-ligation-mediated PCR (CLM-PCR) in HBV DNA integration sites identification.

METHODSOne HCC biopsy was obtained from surgically resected sample. The patient was positive for serum hepatitis B surface antigen (HBsAg). The genomic DNA was purified by the standard phenol/chloroform extraction and ethanol precipitation method. Seperated set of primers were designed to amplify the HBV DNA integration region by means of 3 different PCR methods respectively. The PCR products were analyzed by electrophoresis, then cloned to PMD18-T vector for DNA sequencing. The sequence alignment was performed under Blast software.

RESULTS7 bands and 22 sequencing results was obtained from IPCR and 3 integration sites was identified. Alu-PCR provided 12 bands and 32 sequencing results, and CLM-PCR showed 12 bands and 4 sequencing results. No integration site was identified from the latter two.

CONCLUSIONIPCR compared with another two methods showed a reliable capacity in HBV DNA integration site identification.

Adult ; Biopsy ; Carcinoma, Hepatocellular ; pathology ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Humans ; Liver Neoplasms ; pathology ; virology ; Male ; Polymerase Chain Reaction ; methods ; Virus Integration

AIMTo study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration.

METHODSSperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes.

RESULTSSpecific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9 (9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant difference among the signal spots. The distribution of signal sites among chromosomes seems to be random.

CONCLUSIONHBV could integrate into human sperm chromosomes. Results suggest that the possibility of vertical transmission of HBV via the germ line to the next generation is present.

Adult ; Chromosomes, Human ; genetics ; virology ; Hepatitis B ; genetics ; transmission ; virology ; Hepatitis B virus ; genetics ; Humans ; In Situ Hybridization, Fluorescence ; Infectious Disease Transmission, Vertical ; Male ; Spermatozoa ; virology ; Virus Integration

OBJECTIVESTo study the phenomena of hepatitis B virus (HBV) integration into the tissues of hilar cholangiocarcinoma (HCCA) and to identify the integration sites in the host genome.

METHODSTen fresh HCCA samples were collected from the tissues by surgical ablation, 1 normal hilar bile duct sample selected as control. Cellular DNA were extracted by Wizard SV Genomic DNA Purification System. PCR-derived assay (HBV-Alu-PCR) was employed to amplify the viral-host junctions which contain the HBV sequence and the adjacent cellular flanking sequences. The PCR products were purified and subjected to sequencing by ABI-3730XL Auto DNA Analyzer. The sequence analysis of viral-host junctions was performed by DNASIS MAX 3.0 bioinformatics software. The insertion sites between viral and cellular sequences were identified through homology comparison using NCBI BLAST and MapViewer search.

RESULTSIn 10 HCCA samples, 5 were demonstrated to have HBV integration fragments with total 6 inserted sites identified. Sequence analysis from viral-host junction showed that HBV X gene inserted into host genome at random distribution with truncated fragments. HBV integration recurrently targeted the unknown region in upstream of CXXC finger protein-1 (CpG-binding protein) gene (4 cases). p53 tumor suppressor gene was also found at the integration site.

CONCLUSIONSThere is high integration rate of HBV DNA into cellular genome of HCCA. HBV integration is found frequently into or close to cancer-related genes. The findings demonstrate that HBV infection might have association with the pathogenesis of HCCA.

Aged ; Base Sequence ; Bile Duct Neoplasms ; genetics ; virology ; Cholangiocarcinoma ; genetics ; virology ; DNA, Viral ; genetics ; Female ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Virus Integration

Carcinoma, Hepatocellular ; virology ; DNA, Viral ; metabolism ; Hepatitis B virus ; genetics ; Hepatitis B, Chronic ; virology ; Humans ; Liver Cirrhosis ; virology ; Liver Neoplasms ; virology ; Virus Integration ; physiology

Four out of 10 patients of X-linked severe combined immunodeficiency (X-SCID) were finally developed leukemia after receiving the treatment of gene therapy delivered by gamma-retroviral vectors. This is due to the vector integrated to the proximity of lmo2 etc proto-oncogene promoters, leading to the activation of onco-gene expression, which raises the concern of the bio-safety of gene therapy vectors. Lentiviral vectors, especially self-inactivating lentiviral vectors, are considered to be much safer than gamma-retroviral vectors. However self-inactivating lentiviral vectors also have encountered with some unsafe factors and one of them is the problem of transcriptional "read-through" . During the past years, achievements have been made to reduce lentiviral vector transcriptional read-through, which are reviewed herein.
Animals ; Genetic Therapy ; adverse effects ; methods ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Promoter Regions, Genetic ; Proto-Oncogene Proteins ; genetics ; Transcription, Genetic ; genetics ; Virus Inactivation ; Virus Integration

OBJECTIVETo investigate the prevalence of physical state of HPV-16 DNA in cervical cancer and cervical precancerous carcinoma.

METHODSMultiplex PCR was adopted to detect the physical state of HPV in samples from 252 patients with cervical carcinoma, including 48 samples of cervical cancer, 204 cervical intraepithelial neoplasia (CIN ) (125 CIN I, 46 CIN II and 33 CIN III) and 20 normal samples from the subjects with hysteromyoma undergoing hysterectomy, respectively.

RESULTSAmong 48 patients with cervical cancer, 31 (65.6%) were infected with HPV-16. Eighteen among 31 (58.1%) HPV-16 infected patients with cervical cancer were found to have integrated infection of HPV-16. The positive rates of HPV-16 infection in the patients with CIN I, CIN II and CIN III were 19.2%, 34.8% and 42.4%, and the integrated infection rates of HPV-16 were 16.7%, 18.8% and 35.7%, respectively. Compared with patients with different grades of CIN, the integrated rate of HPV-16 infection in those with cervical cancer was significantly elevated.

CONCLUSIONAmong the patients with HPV-16 infection, the integrated state of HPV-16 is positively correlated with the severity of cervical lesions. Combined HPV typing test and detection of integrated viral state contribute to predicting the prognosis of patients with cervical precancerous lesions and increasing the accuracy of screening cervical cancer on the basis of HPV DNA detection.

Cervical Intraepithelial Neoplasia ; virology ; DNA, Viral ; Early Detection of Cancer ; Female ; Human papillomavirus 16 ; physiology ; Humans ; Papillomavirus Infections ; virology ; Uterine Cervical Neoplasms ; virology ; Virus Integration

Female ; Human papillomavirus 18 ; physiology ; Humans ; Papillomavirus Infections ; complications ; Risk Factors ; Uterine Cervical Neoplasms ; epidemiology ; etiology ; pathology ; virology ; Virus Integration ; genetics ; physiology

Animals ; DNA, Viral ; blood ; Ethanol ; pharmacology ; Hepatitis B ; metabolism ; virology ; Hepatitis B virus ; genetics ; physiology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; Virus Integration ; drug effects ; Virus Replication ; drug effects

To identify the integration sites in the host genome for the hepatitis B virus (HBV)-encoded X protein (HBx) in hepatocellular carcinoma (HCC) biopsies that are positive for hepatitis B surface antigen (HBsAg). HCC biopsies were obtained from six patients that were HBV carriers, as demonstrated by the presence of HBsAg in their serum and sero-negativity for antibody to HBsAg. DNA was extracted from the tissue, fractionated, and circularized. Primers were designed according to the HBx sequence and used to amplify the circularized DNA templates by inverse polymerase chain reaction (IPCR). The amplified DNA fragments were checked by electrophoresis, cloned into the PMD18-T expression vector, and sequenced. Sequence alignment was performed by the Blast algorithms. Seven electrophoresis bands yielded 22 sequencing results, which represented a total of three HBx integration sites in the host genome: 19q12, 2q32.2, 22q12. The 19q12 integration site encompasses the CCNE1 gene, which encodes a G1/S-specific cyclin-E1. HBx-related integration sites exist in HBsAg-positive HCC biopsies. The CCNE1 gene may play a role in the development of HBx-related HCC.
Carcinoma, Hepatocellular ; blood ; genetics ; Cyclin E ; genetics ; DNA Primers ; DNA, Viral ; genetics ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; blood ; genetics ; Oncogene Proteins ; genetics ; Trans-Activators ; genetics ; Virus Integration